User:Ramiro Barrantes/FpgNeiRepair
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| - | + | __NOTOC__ | |
| - | == Background on DNA Repair== | + | = The FpgNei Protein Family = |
| + | |||
| + | [[#Background on DNA Repair|1. Background on DNA Repair]] <br> | ||
| + | [[#Background on Fpg/Nei|2. Background on Fpg/Nei]]<br> | ||
| + | [[#Overall Function and Structure|2.1 Overall Function and Structure]]<br> | ||
| + | [[#Latent Structural Characters (LSCs)|3. Latent Structural Characters Summary]]<br> | ||
| + | [[#References|5. References]] | ||
| + | |||
| + | ==Background on DNA Repair== | ||
The genome of any living organism is continuously affected by exogenous and endogenous agents, such as ultraviolet light, ionizing radiation, different chemicals and the cell's own metabolites (such as reactive oxygen). Therefore, different systems have evolved to repair these damages, with some of these systems shared throughout all lifeforms. Therefore, the proper functioning of DNA repair is critical for survival. There are six pathways of DNA repair (reviewed in Friedberg et al), which include base-excision repair. The latter's distinguishing feature is that it removes lesions as single bases, as opposed to dNMPs or short oligonucleotides like other systems. <ref>PMID:18259689</ref> | The genome of any living organism is continuously affected by exogenous and endogenous agents, such as ultraviolet light, ionizing radiation, different chemicals and the cell's own metabolites (such as reactive oxygen). Therefore, different systems have evolved to repair these damages, with some of these systems shared throughout all lifeforms. Therefore, the proper functioning of DNA repair is critical for survival. There are six pathways of DNA repair (reviewed in Friedberg et al), which include base-excision repair. The latter's distinguishing feature is that it removes lesions as single bases, as opposed to dNMPs or short oligonucleotides like other systems. <ref>PMID:18259689</ref> | ||
Base excision repair's signature enzyme are the DNA glycosylases. These enzymes work by recognizing a damaged base, and then hydrolizing the N-glycosidic bond of the damaged deoxynucleoside and thus removing a single damaged base from DNA. The subsequent steps of the pathway (strand incision, gap-filling and ligation) are done by other enzymes. <ref>PMID: 18259689</ref><ref>PMID:19153658</ref> | Base excision repair's signature enzyme are the DNA glycosylases. These enzymes work by recognizing a damaged base, and then hydrolizing the N-glycosidic bond of the damaged deoxynucleoside and thus removing a single damaged base from DNA. The subsequent steps of the pathway (strand incision, gap-filling and ligation) are done by other enzymes. <ref>PMID: 18259689</ref><ref>PMID:19153658</ref> | ||
| - | == Background on Fpg Nei == | + | ==Background on Fpg Nei== |
| - | == Overall Function and Structure == | + | ==Overall Function and Structure== |
{| valign="top" | {| valign="top" | ||
| - | | (For FPG, the structure used was [[1r2y]] and for Nei [[1k3w]]) Members of this family have <scene name='User:Ramiro_Barrantes/Workbench/Gstfpglabeleddomains/ | + | | valign="top"|(For FPG, the structure used was [[1r2y]] and for Nei [[1k3w]]) Members of this family have <scene name='User:Ramiro_Barrantes/Workbench/Gstfpglabeleddomains/4' target="function"> two domains (blue and orange) connected by a hinge region (purple). DNA (gray) binds in a cleft between the domains. </scene>. When enzyme binds to DNA, the damaged base |
| - | <scene name='User:Ramiro_Barrantes/Workbench/ | + | |
| + | <scene name='User:Ramiro_Barrantes/Workbench/Evertedbaseonbstfpg/1' target="function"> is everted</scene>, with <scene name='User:Ramiro_Barrantes/Workbench/Gstfpgintercalationloop/2' target="function"> several residues intercalating in its place</scene>. This superfamily is also characterized by containing a <scene name='User:Ramiro_Barrantes/Workbench/Gstfpgh2th/2' target="function">helix-two-turn-helix motif (H2TH, in purple) which contacts DNA via Asn174 (in yellow)</scene>; as well as a | ||
<scene name='User:Ramiro_Barrantes/Workbench/Gstfpgzincfinger/5' target="function"> zinc finger, which contacts DNA via Arg264 </scene>. Both the H2TH motif and the Zinc finger, as well as other residues | <scene name='User:Ramiro_Barrantes/Workbench/Gstfpgzincfinger/5' target="function"> zinc finger, which contacts DNA via Arg264 </scene>. Both the H2TH motif and the Zinc finger, as well as other residues | ||
<scene name='User:Ramiro_Barrantes/Workbench/Gstfpgdamagestabilizers/1' target="function"> stabilize the damaged base </scene>. <ref>PMID:11912217</ref>. Catalysis is believed to be mediated by <scene name='User:Ramiro_Barrantes/Workbench/Gstfpgcatalyticresidues/3' target="function">P2, E3, E6 and R264</scene>. For information on the mechanism please consult <ref>PMID:16243784</ref><ref>PMID:11847126</ref><ref>PMID:15249553</ref><ref>PMID:11912217</ref><ref>PMID:10921868</ref>. | <scene name='User:Ramiro_Barrantes/Workbench/Gstfpgdamagestabilizers/1' target="function"> stabilize the damaged base </scene>. <ref>PMID:11912217</ref>. Catalysis is believed to be mediated by <scene name='User:Ramiro_Barrantes/Workbench/Gstfpgcatalyticresidues/3' target="function">P2, E3, E6 and R264</scene>. For information on the mechanism please consult <ref>PMID:16243784</ref><ref>PMID:11847126</ref><ref>PMID:15249553</ref><ref>PMID:11912217</ref><ref>PMID:10921868</ref>. | ||
| - | Similarly In EcoNei (structure [[1k3w]], <scene name='User:Ramiro_Barrantes/Workbench/Econeiorientationandkinking/ | + | Similarly In EcoNei (structure [[1k3w]], <scene name='User:Ramiro_Barrantes/Workbench/Econeiorientationandkinking/4' target="function">E2,K52,N168 and R252 provide orientation and kinking to the DNA,</scene>, and <scene name='User:Ramiro_Barrantes/Workbench/Econeistabilizingsites/2' target="function">P1, E5 and R212 and others stabilize the damaged base </scene>(Thymine Glycol in the case of Nei). <ref>PMID:11847126</ref>. Some of these amino acids are stabilized by [a <scene name='User:Ramiro_Barrantes/Workbench/Econeizincfinger/1' target="function"> Zinc Finger </scene> (although a zincless finger motif is present in some of these subfamilies <ref>PMID:15232006</ref>). Analogously to Fpg, <scene name='User:Ramiro_Barrantes/Workbench/Econeiintercalationloop/4'>''E. coli Nei'' residues insert into the vacated spot left by the "flipped" base</scene>), Note that the last two elements discussed, the zinc vs. zincless finger, and the two kinds of intercalation loops, are examples of coevolving functional clusters, groups of amino acids that perform a function, and that might be unnecessary or compensated for within the other subfamilies. We developed a novel method for identifying these clusters and have applied it to bring insight into the structure, function and evolution FpgNei family. |
| - | | <applet load='1K3W' size='500' frame='true' align='left' caption='See structure column for structure reference' name="function" scene='User:Ramiro_Barrantes/Workbench/ | + | | <applet load='1K3W' size='500' frame='true' align='left' caption='See structure column for structure reference' name="function" scene='User:Ramiro_Barrantes/Workbench/Gstfpglabeleddomains/3' /> |
|- | |- | ||
| - | | [[Image:Picture 9.png|thumb|400px|left|Cartoon phylogenetic tree of the FpgNei protein family. Note that this phylogeny can be appreciated in two levels: by the distribution and number of FpgNei subfamilies in different organisms; and by the kinds of damages that can be repaired]] | + | | valign="top"|===Phylogeny=== [[Image:Picture 9.png|thumb|400px|left|Cartoon phylogenetic tree of the FpgNei protein family. Note that this phylogeny can be appreciated in two levels: by the distribution and number of FpgNei subfamilies in different organisms; and by the kinds of damages that can be repaired (see table on the right)]] |
| - | | | + | | valign="top"|===Table of preferred substrates=== |
{| class="wikitable" style="text-align:center" | {| class="wikitable" style="text-align:center" | ||
|- | |- | ||
| Line 36: | Line 45: | ||
| Plant&Fungi || Sp,Gh || <ref>PMID:19217358</ref> | | Plant&Fungi || Sp,Gh || <ref>PMID:19217358</ref> | ||
|- | |- | ||
| - | | Neil1 || Sp,Gh,on double&single strand DNA|| <ref>PMID:15533836</ref> | + | | Neil1 || Sp,Gh,on double&single strand DNA, Both stereoisomers of Tg|| <ref>PMID:15533836</ref><ref>PMID:12713815</ref> |
| - | |- | + | |- |
| Neil2 || Sp,Gh,on double&single stranded DNA || <ref>PMID:15533836</ref> | | Neil2 || Sp,Gh,on double&single stranded DNA || <ref>PMID:15533836</ref> | ||
|- | |- | ||
| Neil3 || not clear || <ref>PMID:19170771</ref> | | Neil3 || not clear || <ref>PMID:19170771</ref> | ||
| + | |} | ||
|} | |} | ||
| + | ===Mechanisms=== | ||
Several authors have suggested mechanisms for these enzymes, please see references for more information <ref>PMID:11847126</ref><ref>PMID:10921868</ref><ref>PMID:16243784</ref><ref>PMID:11912217</ref>. | Several authors have suggested mechanisms for these enzymes, please see references for more information <ref>PMID:11847126</ref><ref>PMID:10921868</ref><ref>PMID:16243784</ref><ref>PMID:11912217</ref>. | ||
| - | Homologous structures have been solved, including Fpg protein from Lactococcus Lactis ([[1pjj]])<ref>PMID: 16243784</ref>, Bacillus Stereothermophilus ([[1r2y]])<ref>PMID:14525999</ref>, Thermos Thermophilus ([[1ee8]])<ref>PMID:10921868</ref> and Escherichia Coli([[1k82]])<ref>PMID:11912217</ref> and Nei from Escherichia Coli ([[1k3w]])<ref>PMID:11847126</ref>. The overall structure is similar, and some of the damages include 8-oxoguanine and fapyG ([[1xc8]])<ref>PMID:15249553</ref>. | + | ===Solved Structures=== |
| + | Homologous structures have been solved, including Fpg protein from Lactococcus Lactis ([[1pjj]])<ref>PMID: 16243784</ref>, Bacillus Stereothermophilus ([[1r2y]])<ref>PMID:14525999</ref>, Thermos Thermophilus ([[1ee8]])<ref>PMID:10921868</ref> and Escherichia Coli([[1k82]])<ref>PMID:11912217</ref> and Nei from Escherichia Coli ([[1k3w]])<ref>PMID:11847126</ref>, as well as human Neil1 ([[1tdh]])<ref>PMID:19625256</ref>and mimivirus Nei ([[3a42]])<ref>PMID:17627905</ref>. The overall structure is similar, and some of the damages include 8-oxoguanine and fapyG ([[1xc8]])<ref>PMID:15249553</ref>. | ||
| + | ==Latent Structural Characters (LSCs)== | ||
| + | {| | ||
| + | | [[Image:LSCDiagram.png|thumb|500px|right|Diagram with the LSCs (yellow), their suggested role (arrows) and the amino acids or features they affect (green and blue)]] We define a latent structural character (LSC) as neighboring amino acids which have changed in rate or constraint for a given clade or set of clades. For example, the cysteines in the zinc finger are all conserved in the fpg1, fpg2, actinobacterial and eukaryotic clades, but have a higher rate in plants and neil1, as the latter have a zinc-less finger (see [http://proteopedia.org/wiki/index.php?title=User:Ramiro_Barrantes/Workbench/LSC6 here] for more information). We developed a novel method to find these groups of amino acids, discovering previously unknown groups of amino acids which are functionally important in some of these subfamilies. | ||
| + | The table below has some of our main findings, there are two variants for each LSC in case that for one LSC there is a compensating cluster in the other subfamilies. If you click on one of them you will see the result in the structure, and you can go to the explanation and the sequence distribution below. <br> [http://xpdb.nist.gov/hivsdb/jmol/jmol_help.html Click here for Jmol tutorial].| | ||
|} | |} | ||
| - | |||
| - | == Latent Structural Characters (LSCs) == | ||
| - | We define a latent structural character (LSC) as neighboring amino acids which have changed in rate or constraint for a given clade or set of clades. For example, the cysteines in the zinc finger are all conserved in the fpg1, fpg2, actinobacterial and eukaryotic clades, but have a higher rate in plants and neil1, as the latter have a zinc-less finger ([[#Functional Cluster: Zinc/zincless finger|see here for more information]]). We developed a novel method to find these groups of amino acids, discovering previously unknown groups of amino acids which are functionally important in some of these subfamilies. | ||
| - | |||
| - | The table below has some of our main findings, there are two variants for each LSC in case that for one LSC there is a compensating cluster in the other subfamilies. If you click on one of them you will see the result in the structure, and you can go to the explanation and the sequence distribution below. <br> | ||
| - | '''Suggestion: Once you have an animation you would like to study, we recommend clicking the "pop-up" button to create a new screen so that you can read the text alongside'''. [http://xpdb.nist.gov/hivsdb/jmol/jmol_help.html Click here for Jmol tutorial]. | ||
| - | |||
| - | |||
| - | |||
| - | === LSC1: Stability of perfectly conserved Asn168 === | ||
| - | {| - style="font-size:15px;align=left;valign=top" | ||
| - | | rowspan="2" |<p>[[Image:F1WebLogo.jpg|thumb|166px|left|WebLogo for the first functional unit, note the equivalent roles between lysine and arginine, which hydrogen-bond with the crucial Asn174]] (<scene name='User:Ramiro_Barrantes/Workbench/Lys160asp178/13' target="lsc1-1"> Click here to watch this cluster in Geobacillus Fpg: present in Fpg1, Fpg2 and Plants and Fungi</scene> and <scene name='User:Ramiro_Barrantes/Workbench/Econeiarginine171/10' target="lsc1-2"> here for the compensating one in E. Coli: present in Actinobacteria 1 and 2, Proteobacteria and all vertebrate subfamilies </scene>). Asn174 (in yellow), part of the helix-two-turn-helix (H2TH) domain (in orange), along with two other amino acids (including the key amino acid Arg264, in yellow) have an effect in the orientation and kinking of the DNA <ref>PMID:11847126</ref>. Both the H2TH and the zinc finger are the two DNA binding motifs of FpgNei <ref>PMID:10921868</ref>. In 4 of the 9 clades (Fpg1, Fpg2 and Plants and Fungi) we hypothesize that Asn174 is stabilized by the amino acid corresponding to Lys160, which in turn hydrogen bonds with Thr266 and Gly265. In GstFpg, Lys160 helps keep the proper arrangement between the zinc finger (in blue) and the H2TH (in beige) <ref>PMID:10921868</ref>. In the other subfamilies (Actinobacteria 1 and 2, Proteobacteria and all vertebrate subfamilies), this role is played by Arg171, which originates on a different helix, and hydrogen bonds to the other beta-sheet of the zinc-finger. Site directed mutagenesis on Lys155 (corresponding lysine in E. coli Fpg) and on Arg171 result in premature dissociation and/or loss of activity <ref>PMID:11554299</ref><ref>PMID:17002303</ref>. Our analysis revealed the possible compensatory role of both LSCs as well as hypotheses on other residues which might be important to stabilize Asn174.</p> | ||
| - | | <applet load='1R2Y' size='380' frame='true' align='right' name="lsc1-1" scene='User:Ramiro_Barrantes/Workbench/Lys160asp178/13'/> | ||
| - | |- | ||
| - | | <applet load='1K3W' size='380' frame='true' align='right' name="lsc1-2" scene='User:Ramiro_Barrantes/Workbench/Econeiarginine171/10' /> | ||
| - | |} | ||
| - | |||
| - | |||
| - | === LSC2: Stability of catalytic helix === | ||
| - | {| - style="font-size:15px;align=left;valign=top" | ||
| - | | rowspan="2" |<p>(<scene name='User:Ramiro_Barrantes/Workbench/Gstfpgglu8arg57leu4triad/8' target="lsc2-1"> Click here to watch this cluster in Geobacillus Fpg: present in Fpg1, Fpg2, Plants and Fungi</scene> and <scene name='User:Ramiro_Barrantes/Workbench/Econeisupporttriadcounterpart/5' target="lsc2-2"> here for how this region looks in ''E. coli'': present in Actinomycetes 1 and 2, Proteobacteria and all vertebrate subfamilies </scene>) [[Image:F2WebLogo.jpg|thumb|166px|left|The LER triad interact and might stabilize the catalytic helix (which includes Pro2 and Glu3 and Glu6), it is not clear how this is achieved in the other subfamilies]] The triad Leu4, Glu8 and Arg57 is highly conserved in fpg1, fpg2, and plants and fungi. However, the role of these group of amino acids has not been discussed in the literature. Site directed mutagenesis has been performed on Arg57, resulting in reduced binding <ref>PMID:16928690</ref> (glycosylase activity was not measured). We entertain two hypotheses about the role of this triad: perhaps this group of amino acids stabilizes helixA (brown), which contains the catalytic residues Pro2,Glu3 and Glu6 <ref>PMID:16243784</ref><ref>PMID:10921868</ref>. Another possible role is in contributing to the stability of Lys60, key for glycosylase and lyase activity, and thus with the other LSC related to the stability of site Lys60. The key interaction for this role corresponds to the highly conserved Glu137 (yellow), which when mutated results in 20-fold decrease in activity in EcoNei<ref>PMID:11106507</ref><ref>PMID:15610004</ref>). However, the corresponding amino acid in ''E. coli'' (D128) does not affect activity after mutated<ref>PMID:11711552</ref>. Please see next LSC for more information. Therefore, our analysis shed light on an unstudied group of cooperating amino acids which might be important for function. </p> | ||
| - | | <applet load='1R2Y' size='380' frame='true' align='right' name="lsc2-1" scene='User:Ramiro_Barrantes/Workbench/Gstfpgglu8arg57leu4triad/8'/> | ||
| - | |- | ||
| - | | <applet load='1K3W' size='380' frame='true' align='right' name="lsc2-2" scene='User:Ramiro_Barrantes/Workbench/Econeisupporttriadcounterpart/5' /> | ||
| - | |} | ||
| - | |||
| - | === LSC3: Neil1-specific: Stability of Lys60 === | ||
| - | {| - style="font-size:15px;align=left;valign=top" | ||
| - | | rowspan="2" |<p>[[Image:Lys60Stability.jpg|thumb|166px|right|Notice the covariation in Neil1 and the rest with positions 134 and 137 and 170]](<scene name='User:Ramiro_Barrantes/Workbench/Gstfpgneil1covariate/5' target="lsc3-1">Click here to look at network of sites stabilizing key residue Lysine 60</scene> and <scene name='User:Ramiro_Barrantes/Workbench/Neil1lys60support/3' target="lsc3-2"> here for the compensating network in Neil1, mainly Asn172</scene>) Gly59 and Lys60 (yellow) are important in the activity of MutM <ref>PMID:9826758</ref><ref>PMID:11847126</ref> (reflected by their perfect conservation). Lys60 interacts with the the 3' phosphate of 8oxoG <ref>PMID:16928690</ref>. Our analysis revealed a site (Glu137) which is the main hydrogen bond with Gly59. Mutation of this site in Fpg results in 20-fold decrease in activity<ref>PMID:11106507</ref><ref>PMID:15610004</ref>. In Fpg, Plants and Fungi, this LSC could be related to the triad Leu4/Glu8/Arg57. Glu/Asp 137 is present in all clades except Neil1, however, in | ||
| - | <scene name='User:Ramiro_Barrantes/Workbench/Econeilys52supportfromasp128/1' target="lsc3-1"> EcoNei it hydrogen bonds with a water molecule </scene>. This amino acid is compensated for by Asn172 in Neil1 (in green). Therefore, Our analysis showed a key amino acid which has been compensated for in a differnet clade for Asn172, however, it came up as part of a network to other important amino acids. It is interesting that Glu137 was replaced by a cysteine (Cys136) in Neil1. </p> | ||
| - | | <applet load='1R2Y' size='380' frame='true' align='right' name="lsc3-1" scene='User:Ramiro_Barrantes/Workbench/Gstfpgneil1covariate/5' /> | ||
| - | |- | ||
| - | | <applet load='1TDH' size='380' frame='true' align='right' name="lsc3-2" scene='User:Ramiro_Barrantes/Workbench/Neil1lys60support/3' /> | ||
| - | |} | ||
| - | |||
| - | === LSC4: Intercalation Loop === | ||
| - | {| - style="font-size:15px;align=left;valign=top" | ||
| - | | rowspan="2" |<p><scene name='User:Ramiro_Barrantes/Workbench/Fpgintercalationloop/3' target="lsc4-1">Click here for intercalation loop in Fpg1, Fpg2 Plants and Fungi, Neil1 and Mimivirus</scene> and <scene name='User:Ramiro_Barrantes/Workbench/Econeiintercalationloop/4' target="lsc4-2">here for corresponding structure in ''E. coli'' Nei</scene>) | ||
| - | [[Image:IntercalationLoopWebLogo.jpg|thumb|166px|right|The residue in positions 77 and 78 suggest a possible intercalation loop]] The intercalation loop (green) inserts into the space left by the excised base. This structure, as well as a group of amino acids that support it (also in green) exhibit high conservation in Fpg, AY, plants and Neil1, whereas the other have a higher substitution rate in the corresponding amino acid, while containing the methionine (yellow). The best studied example is its counterpart is in ''E. coli'' Nei, which fulfills the same purpose as the Fpg intercalation loop <ref>PMID:17002303</ref>, by inserting into the DNA when the base is everted. Note that the amino acid composition suggests that mimivirus has this structure.</p> | ||
| - | | <applet load='1R2Y' size='380' frame='true' align='right' name="lsc4-1" scene='User:Ramiro_Barrantes/Workbench/Fpgintercalationloop/3' /> | ||
| - | |- | ||
| - | | <applet load='1K3W' size='380' frame='true' align='right' name="lsc4-2" scene='User:Ramiro_Barrantes/Workbench/Econeiintercalationloop/4' /> | ||
| - | |} | ||
| - | |||
| - | === LSC5: DNA Binding Tyrosine === | ||
| - | {| - style="font-size:15px;align=left;valign=top" | ||
| - | | <p>[[Image:PlantSpecific.jpg|thumb|166px|right|Tyrosine that binds to DNA, plants have a different structure alltogether]] | ||
| - | <scene name='User:Ramiro_Barrantes/Workbench/Gstfpgtyr243/3' target="lsc5-1">(Click here to observe this network on the BstFpg structure)</scene> | ||
| - | This tyrosine hydrogen bonds with Glu179 (BstFpg coordinates). Glu179 is perfectly conserved throughout all clades, and its mutation results in loss of activity <ref>PMID:11106507</ref>. We find that this Tyrosine is replaced by an arginine in plants, however, this arginine could be fulfilling the same role as the tyrosine and might not account for any difference in function. This is consistent with Glu179 and Tyr243 being part of the same network and for Tyr243 playing an important role in the function of the protein. However, this amino acid hasn't been studied. On the other hand, Arg244 is very hightly conserved in everything but Neil1 and plants, consistent with the idea that it might contribute to Zinc Finger stability. The presence of Arg244 correlates with the presence of Pro248, which is the amino acid right before Cys249, which holds the Zinc. </p> | ||
| - | | <applet load='1R2Y' size='380' frame='true' align='right' name="lsc5-1" scene='User:Ramiro_Barrantes/Workbench/Gstfpgtyr243/4' /> | ||
| - | |} | ||
| - | |||
| - | === LSC6: Zinc/zincless finger === | ||
| - | {| - style="font-size:15px;align=left;valign=top" | ||
| - | | rowspan="2" |<p>[[Image:ZincFinger.jpg|thumb|166px|right|The Zinc Finger helps as a support of the key R264 residue]]<scene name='User:Ramiro_Barrantes/Workbench/Gstfpgzincfinger/6' target="lsc6-1"> Click here to see the Zinc finger on Bst Fpg, note the 4 cysteines. The zinc finger is key for DNA binding </scene> | ||
| - | and <scene name='User:Ramiro_Barrantes/Workbench/Neil1zinclessfinger/5' target="lsc6-2"> click here for the Neil1 zincless finger, an equivalent structure without the zinc nor the cysteines.</scene></p> | ||
| - | <p>The Zinc finger (of the β/β-antiparallel CCCC type) serves to hold the absolutely conserved Arg264 residue <ref>PMID:11847126</ref>, which binds to the phosphate of the damaged base and is crucial for function (mutation of this site results in failed cleavage of the damaged base <ref>PMID:11847126</ref>). Site directed mutagenesis experiments have been performed on all four cysteines, leading to loss of activity <ref>PMID:8473347</ref><ref>PMID: 8253809 </ref>, emphasizing the importance of this LSC. In Neil1, there is no Zinc but there is an equivalent structure: a "zincless finger" <ref>PMID:15232006</ref>. Both the plants and mimivirus have a zincless finger as well <ref>PMID:19217358</ref><ref>PMID:17627905</ref>, although it is not clear if these are all homologous.</p> | ||
| - | | <applet load='1R2Y' size='380' frame='true' align='right' name="lsc6-1" scene='User:Ramiro_Barrantes/Workbench/Gstfpgzincfinger/6' /> | ||
| - | |- | ||
| - | | <applet load='1TDH' size='380' frame='true' align='right' name="lsc6-2" scene='User:Ramiro_Barrantes/Workbench/Neil1zinclessfinger/5' /> | ||
| - | |} | ||
| - | |||
| - | |||
{| - style="font-size:18px;align=center" | {| - style="font-size:18px;align=center" | ||
| Line 119: | Line 72: | ||
{| class="wikitable" style="text-align:center" | {| class="wikitable" style="text-align:center" | ||
|- | |- | ||
| - | ! Functional Cluster !! | + | ! Functional Cluster !! State 1 !! State 2 !! Fpg1 !! Fpg2 !! Plant !! Neil1 !! Neil2 !! Neil3 !! Proteo !! Actino1 !! Actino2 !! MimiVirus |
|- | |- | ||
| - | | Support for perfectly conserved | + | | Support for perfectly conserved residue |
| <scene name='User:Ramiro_Barrantes/Workbench/Lys160asp178/13' target="fpg">Lys160 and Asp178</scene> | | <scene name='User:Ramiro_Barrantes/Workbench/Lys160asp178/13' target="fpg">Lys160 and Asp178</scene> | ||
| - | | <scene name='User:Ramiro_Barrantes/Workbench/Econeiarginine171/ | + | | <scene name='User:Ramiro_Barrantes/Workbench/Econeiarginine171/10' target="ecoNei">Arg171</scene> |
| 1 || 1 || 1 || 0 || 0 || 0 || 0 || 0 || 0 || 0 | | 1 || 1 || 1 || 0 || 0 || 0 || 0 || 0 || 0 || 0 | ||
|- | |- | ||
| Stability of catalytic helix | | Stability of catalytic helix | ||
| - | | <scene name='User:Ramiro_Barrantes/Workbench/Gstfpgglu8arg57leu4triad/ | + | | <scene name='User:Ramiro_Barrantes/Workbench/Gstfpgglu8arg57leu4triad/8' target="fpg">Leu4,Glu8,Arg57</scene> |
| - | | <scene name='User:Ramiro_Barrantes/Workbench/Econeisupporttriadcounterpart/ | + | | <scene name='User:Ramiro_Barrantes/Workbench/Econeisupporttriadcounterpart/5' target="ecoNei">Different</scene> |
| 1 || 1 || 1 || - || - || - || - || - || - || - | | 1 || 1 || 1 || - || - || - || - || - || - || - | ||
|- | |- | ||
| Neil1-specific: Stability of Lys60 | | Neil1-specific: Stability of Lys60 | ||
| - | | <scene name='User:Ramiro_Barrantes/Workbench/Gstfpgneil1covariate/ | + | | <scene name='User:Ramiro_Barrantes/Workbench/Gstfpgneil1covariate/5' target="fpg">E137,R58,G135,L134</scene> |
| - | | <scene name='User:Ramiro_Barrantes/Workbench/Neil1lys60support/ | + | | <scene name='User:Ramiro_Barrantes/Workbench/Neil1lys60support/3' target="ecoNei">Covariation with Asn172</scene> |
| - | | | + | | 1 || 1 || 1|| 0 || 1 || 1 || 1 || 1 || 1 || 1 |
|- | |- | ||
| Intercalation loop | | Intercalation loop | ||
| - | | <scene name='User:Ramiro_Barrantes/Workbench/Fpgintercalationloop/ | + | | <scene name='User:Ramiro_Barrantes/Workbench/Fpgintercalationloop/3' target="fpg">D110,F108,R113,R112,F114</scene> |
| - | | <scene name='User:Ramiro_Barrantes/Workbench/Econeiintercalationloop/ | + | | <scene name='User:Ramiro_Barrantes/Workbench/Econeiintercalationloop/4' target="ecoNei">Gln76,Met77,Tyr78</scene> |
| - | | | + | | 1 || 1 || 1 || - || - || - || - || - || - || 1 |
|- | |- | ||
| PlantFungi-specific: R254 DNA binding | | PlantFungi-specific: R254 DNA binding | ||
| - | | <scene name='User:Ramiro_Barrantes/Workbench/Gstfpgtyr243/ | + | | <scene name='User:Ramiro_Barrantes/Workbench/Gstfpgtyr243/4' target="fpg">Y242,G243,R244</scene> |
| different in plants | | different in plants | ||
| - | | | + | | 1 || 1 || 0 || 1 || 1 || 1 || 2 || 1 || 1 || 1 |
|- | |- | ||
| - | | | + | | Zinc/zincless finger |
| - | | <scene name='User:Ramiro_Barrantes/Workbench/Gstfpgzincfinger/ | + | | <scene name='User:Ramiro_Barrantes/Workbench/Gstfpgzincfinger/6' target="fpg">Zinc finger</scene> |
| - | | <scene name='User:Ramiro_Barrantes/Workbench/Neil1zinclessfinger/ | + | | <scene name='User:Ramiro_Barrantes/Workbench/Neil1zinclessfinger/5' target="ecoNei">Neil1 zincless finger</scene> |
| - | | | + | | 1 || 1 || - || - || 1 || 1 || 1 || 1 || 1 || - |
|- | |- | ||
| Recognition complex | | Recognition complex | ||
| <scene name='User:Ramiro_Barrantes/Workbench/Gstfpgrecognitioncomplex/1' target="fpg">Loop from Gly218 to Gly233</scene> | | <scene name='User:Ramiro_Barrantes/Workbench/Gstfpgrecognitioncomplex/1' target="fpg">Loop from Gly218 to Gly233</scene> | ||
| - | | | + | | <scene name='User:Ramiro_Barrantes/Workbench/LSCs/Neil1helixalpha10beta9/1' target="ecoNei">Helix in Neil1</scene> |
| - | | | + | | 1 || - || - || - || - || - || - || - || - || - |
|} | |} | ||
| - | |||
| - | <table><tr valign="top"><td> | ||
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| - | |||
| - | </td></tr> | ||
| - | <tr valign="top"><td> | ||
| - | |||
| - | === LSC7: Recognition Complex === | ||
| - | This complex is key in recognizing damaged guanine, in the literature it is referred to as the alpha10-b9 loop. For details and hypothesis on the structural basis of 8oxoG and fapyG recognition please refer to <ref>PMID:14525999</ref><ref>PMID:15249553</ref>. The area corresponding to this complex is much shorter in some of the other subfamilies, and there is no obvious homology. In the EcoNei and Neil1 structures this loop is absent as well <ref>PMID:15232006</ref><ref>PMID:11847126</ref>. We showed that the distribution of the rates of substitution within this loop is non-random, providing further support for the importance of the loop in the specificity in Fpg. This non-randomness is also present in 3 of the other subfamilies perhaps suggesting a possible important role of this area in those subfamilies. | ||
| - | </td> | ||
| - | <td> | ||
| - | </td> | ||
| - | </tr> | ||
| - | </table> | ||
| - | |||
| - | == Zinc/zincless finger == | ||
| - | [[Image:ZincFingerComparison.png|thumb|166px|right|Tyrosine that binds to DNA, the plant has a different structure alltogether]] | ||
== Evolution == | == Evolution == | ||
The FpgNei evolution has not been easy to resolve <ref>PMID:15232006</ref>, especially in the deeper branches. Assuming that functional clusters evolve more slowly than individual residues, we can use this as phylogenetic characters to 1) draw the most parsimonious evolution of the superfamily as dictated by these functional clusters 2) examine how these clusters have evolved and how this might have influenced the evolution of FpgNei. | The FpgNei evolution has not been easy to resolve <ref>PMID:15232006</ref>, especially in the deeper branches. Assuming that functional clusters evolve more slowly than individual residues, we can use this as phylogenetic characters to 1) draw the most parsimonious evolution of the superfamily as dictated by these functional clusters 2) examine how these clusters have evolved and how this might have influenced the evolution of FpgNei. | ||
| - | [[Image:Scenario1.png | + | [[Image:Scenario1.png|thumb|500px|right|Gene evolution of the Fpg/Nei protein family. The root is placed in the Fpg branch given its broad distribution among bacteria.)]] |
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==References== | ==References== | ||
<references /> | <references /> | ||
Current revision
The FpgNei Protein Family
1. Background on DNA Repair
2. Background on Fpg/Nei
2.1 Overall Function and Structure
3. Latent Structural Characters Summary
5. References
Background on DNA Repair
The genome of any living organism is continuously affected by exogenous and endogenous agents, such as ultraviolet light, ionizing radiation, different chemicals and the cell's own metabolites (such as reactive oxygen). Therefore, different systems have evolved to repair these damages, with some of these systems shared throughout all lifeforms. Therefore, the proper functioning of DNA repair is critical for survival. There are six pathways of DNA repair (reviewed in Friedberg et al), which include base-excision repair. The latter's distinguishing feature is that it removes lesions as single bases, as opposed to dNMPs or short oligonucleotides like other systems. [1]
Base excision repair's signature enzyme are the DNA glycosylases. These enzymes work by recognizing a damaged base, and then hydrolizing the N-glycosidic bond of the damaged deoxynucleoside and thus removing a single damaged base from DNA. The subsequent steps of the pathway (strand incision, gap-filling and ligation) are done by other enzymes. [2][3]
Background on Fpg Nei
Overall Function and Structure
| (For FPG, the structure used was 1r2y and for Nei 1k3w) Members of this family have . When enzyme binds to DNA, the damaged base
, with . This superfamily is also characterized by containing a ; as well as a . Both the H2TH motif and the Zinc finger, as well as other residues . [4]. Catalysis is believed to be mediated by . For information on the mechanism please consult [5][6][7][8][9]. Similarly In EcoNei (structure 1k3w, , and (Thymine Glycol in the case of Nei). [10]. Some of these amino acids are stabilized by [a (although a zincless finger motif is present in some of these subfamilies [11]). Analogously to Fpg, ), Note that the last two elements discussed, the zinc vs. zincless finger, and the two kinds of intercalation loops, are examples of coevolving functional clusters, groups of amino acids that perform a function, and that might be unnecessary or compensated for within the other subfamilies. We developed a novel method for identifying these clusters and have applied it to bring insight into the structure, function and evolution FpgNei family. |
| ||||||||||||||||||||||||||||||
===Phylogeny===  | ===Table of preferred substrates===
|
Mechanisms
Several authors have suggested mechanisms for these enzymes, please see references for more information [21][22][23][24].
Solved Structures
Homologous structures have been solved, including Fpg protein from Lactococcus Lactis (1pjj)[25], Bacillus Stereothermophilus (1r2y)[26], Thermos Thermophilus (1ee8)[27] and Escherichia Coli(1k82)[28] and Nei from Escherichia Coli (1k3w)[29], as well as human Neil1 (1tdh)[30]and mimivirus Nei (3a42)[31]. The overall structure is similar, and some of the damages include 8-oxoguanine and fapyG (1xc8)[32].
Latent Structural Characters (LSCs)
 The table below has some of our main findings, there are two variants for each LSC in case that for one LSC there is a compensating cluster in the other subfamilies. If you click on one of them you will see the result in the structure, and you can go to the explanation and the sequence distribution below. |
|
|
| Functional Cluster | State 1 | State 2 | Fpg1 | Fpg2 | Plant | Neil1 | Neil2 | Neil3 | Proteo | Actino1 | Actino2 | MimiVirus |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Support for perfectly conserved residue | 1 | 1 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | ||
| Stability of catalytic helix | 1 | 1 | 1 | - | - | - | - | - | - | - | ||
| Neil1-specific: Stability of Lys60 | 1 | 1 | 1 | 0 | 1 | 1 | 1 | 1 | 1 | 1 | ||
| Intercalation loop | 1 | 1 | 1 | - | - | - | - | - | - | 1 | ||
| PlantFungi-specific: R254 DNA binding | different in plants | 1 | 1 | 0 | 1 | 1 | 1 | 2 | 1 | 1 | 1 | |
| Zinc/zincless finger | 1 | 1 | - | - | 1 | 1 | 1 | 1 | 1 | - | ||
| Recognition complex | 1 | - | - | - | - | - | - | - | - | - |
Evolution
The FpgNei evolution has not been easy to resolve [33], especially in the deeper branches. Assuming that functional clusters evolve more slowly than individual residues, we can use this as phylogenetic characters to 1) draw the most parsimonious evolution of the superfamily as dictated by these functional clusters 2) examine how these clusters have evolved and how this might have influenced the evolution of FpgNei.
References
- ↑ Zharkov DO. Base excision DNA repair. Cell Mol Life Sci. 2008 May;65(10):1544-65. PMID:18259689 doi:10.1007/s00018-008-7543-2
- ↑ Zharkov DO. Base excision DNA repair. Cell Mol Life Sci. 2008 May;65(10):1544-65. PMID:18259689 doi:10.1007/s00018-008-7543-2
- ↑ Robertson AB, Klungland A, Rognes T, Leiros I. DNA repair in mammalian cells: Base excision repair: the long and short of it. Cell Mol Life Sci. 2009 Mar;66(6):981-93. PMID:19153658 doi:10.1007/s00018-009-8736-z
- ↑ Gilboa R, Zharkov DO, Golan G, Fernandes AS, Gerchman SE, Matz E, Kycia JH, Grollman AP, Shoham G. Structure of formamidopyrimidine-DNA glycosylase covalently complexed to DNA. J Biol Chem. 2002 May 31;277(22):19811-6. Epub 2002 Mar 23. PMID:11912217 doi:http://dx.doi.org/10.1074/jbc.M202058200
- ↑ Pereira de Jesus K, Serre L, Zelwer C, Castaing B. Structural insights into abasic site for Fpg specific binding and catalysis: comparative high-resolution crystallographic studies of Fpg bound to various models of abasic site analogues-containing DNA. Nucleic Acids Res. 2005 Oct 20;33(18):5936-44. Print 2005. PMID:16243784 doi:http://dx.doi.org/33/18/5936
- ↑ Zharkov DO, Golan G, Gilboa R, Fernandes AS, Gerchman SE, Kycia JH, Rieger RA, Grollman AP, Shoham G. Structural analysis of an Escherichia coli endonuclease VIII covalent reaction intermediate. EMBO J. 2002 Feb 15;21(4):789-800. PMID:11847126 doi:10.1093/emboj/21.4.789
- ↑ Coste F, Ober M, Carell T, Boiteux S, Zelwer C, Castaing B. Structural basis for the recognition of the FapydG lesion (2,6-diamino-4-hydroxy-5-formamidopyrimidine) by formamidopyrimidine-DNA glycosylase. J Biol Chem. 2004 Oct 15;279(42):44074-83. Epub 2004 Jul 10. PMID:15249553 doi:10.1074/jbc.M405928200
- ↑ Gilboa R, Zharkov DO, Golan G, Fernandes AS, Gerchman SE, Matz E, Kycia JH, Grollman AP, Shoham G. Structure of formamidopyrimidine-DNA glycosylase covalently complexed to DNA. J Biol Chem. 2002 May 31;277(22):19811-6. Epub 2002 Mar 23. PMID:11912217 doi:http://dx.doi.org/10.1074/jbc.M202058200
- ↑ Sugahara M, Mikawa T, Kumasaka T, Yamamoto M, Kato R, Fukuyama K, Inoue Y, Kuramitsu S. Crystal structure of a repair enzyme of oxidatively damaged DNA, MutM (Fpg), from an extreme thermophile, Thermus thermophilus HB8. EMBO J. 2000 Aug 1;19(15):3857-69. PMID:10921868 doi:http://dx.doi.org/10.1093/emboj/19.15.3857
- ↑ Zharkov DO, Golan G, Gilboa R, Fernandes AS, Gerchman SE, Kycia JH, Rieger RA, Grollman AP, Shoham G. Structural analysis of an Escherichia coli endonuclease VIII covalent reaction intermediate. EMBO J. 2002 Feb 15;21(4):789-800. PMID:11847126 doi:10.1093/emboj/21.4.789
- ↑ Doublie S, Bandaru V, Bond JP, Wallace SS. The crystal structure of human endonuclease VIII-like 1 (NEIL1) reveals a zincless finger motif required for glycosylase activity. Proc Natl Acad Sci U S A. 2004 Jul 13;101(28):10284-9. Epub 2004 Jul 1. PMID:15232006 doi:10.1073/pnas.0402051101
- ↑ Leipold MD, Muller JG, Burrows CJ, David SS. Removal of hydantoin products of 8-oxoguanine oxidation by the Escherichia coli DNA repair enzyme, FPG. Biochemistry. 2000 Dec 5;39(48):14984-92. PMID:11101315
- ↑ David SS, Williams SD. Chemistry of Glycosylases and Endonucleases Involved in Base-Excision Repair. Chem Rev. 1998 May 7;98(3):1221-1262. PMID:11848931
- ↑ Jiang D, Hatahet Z, Melamede RJ, Kow YW, Wallace SS. Characterization of Escherichia coli endonuclease VIII. J Biol Chem. 1997 Dec 19;272(51):32230-9. PMID:9405426
- ↑ Sidorenko VS, Rot MA, Filipenko ML, Nevinsky GA, Zharkov DO. Novel DNA glycosylases from Mycobacterium tuberculosis. Biochemistry (Mosc). 2008 Apr;73(4):442-50. PMID:18457574
- ↑ Kathe SD, Barrantes-Reynolds R, Jaruga P, Newton MR, Burrows CJ, Bandaru V, Dizdaroglu M, Bond JP, Wallace SS. Plant and fungal Fpg homologs are formamidopyrimidine DNA glycosylases but not 8-oxoguanine DNA glycosylases. DNA Repair (Amst). 2009 May 1;8(5):643-53. Epub 2009 Feb 12. PMID:19217358 doi:10.1016/j.dnarep.2008.12.013
- ↑ Hailer MK, Slade PG, Martin BD, Rosenquist TA, Sugden KD. Recognition of the oxidized lesions spiroiminodihydantoin and guanidinohydantoin in DNA by the mammalian base excision repair glycosylases NEIL1 and NEIL2. DNA Repair (Amst). 2005 Jan 2;4(1):41-50. PMID:15533836 doi:10.1016/j.dnarep.2004.07.006
- ↑ Rosenquist TA, Zaika E, Fernandes AS, Zharkov DO, Miller H, Grollman AP. The novel DNA glycosylase, NEIL1, protects mammalian cells from radiation-mediated cell death. DNA Repair (Amst). 2003 May 13;2(5):581-91. PMID:12713815
- ↑ Hailer MK, Slade PG, Martin BD, Rosenquist TA, Sugden KD. Recognition of the oxidized lesions spiroiminodihydantoin and guanidinohydantoin in DNA by the mammalian base excision repair glycosylases NEIL1 and NEIL2. DNA Repair (Amst). 2005 Jan 2;4(1):41-50. PMID:15533836 doi:10.1016/j.dnarep.2004.07.006
- ↑ Takao M, Oohata Y, Kitadokoro K, Kobayashi K, Iwai S, Yasui A, Yonei S, Zhang QM. Human Nei-like protein NEIL3 has AP lyase activity specific for single-stranded DNA and confers oxidative stress resistance in Escherichia coli mutant. Genes Cells. 2009 Feb;14(2):261-70. Epub 2008 Jan 15. PMID:19170771 doi:10.1111/j.1365-2443.2008.01271.x
- ↑ Zharkov DO, Golan G, Gilboa R, Fernandes AS, Gerchman SE, Kycia JH, Rieger RA, Grollman AP, Shoham G. Structural analysis of an Escherichia coli endonuclease VIII covalent reaction intermediate. EMBO J. 2002 Feb 15;21(4):789-800. PMID:11847126 doi:10.1093/emboj/21.4.789
- ↑ Sugahara M, Mikawa T, Kumasaka T, Yamamoto M, Kato R, Fukuyama K, Inoue Y, Kuramitsu S. Crystal structure of a repair enzyme of oxidatively damaged DNA, MutM (Fpg), from an extreme thermophile, Thermus thermophilus HB8. EMBO J. 2000 Aug 1;19(15):3857-69. PMID:10921868 doi:http://dx.doi.org/10.1093/emboj/19.15.3857
- ↑ Pereira de Jesus K, Serre L, Zelwer C, Castaing B. Structural insights into abasic site for Fpg specific binding and catalysis: comparative high-resolution crystallographic studies of Fpg bound to various models of abasic site analogues-containing DNA. Nucleic Acids Res. 2005 Oct 20;33(18):5936-44. Print 2005. PMID:16243784 doi:http://dx.doi.org/33/18/5936
- ↑ Gilboa R, Zharkov DO, Golan G, Fernandes AS, Gerchman SE, Matz E, Kycia JH, Grollman AP, Shoham G. Structure of formamidopyrimidine-DNA glycosylase covalently complexed to DNA. J Biol Chem. 2002 May 31;277(22):19811-6. Epub 2002 Mar 23. PMID:11912217 doi:http://dx.doi.org/10.1074/jbc.M202058200
- ↑ Pereira de Jesus K, Serre L, Zelwer C, Castaing B. Structural insights into abasic site for Fpg specific binding and catalysis: comparative high-resolution crystallographic studies of Fpg bound to various models of abasic site analogues-containing DNA. Nucleic Acids Res. 2005 Oct 20;33(18):5936-44. Print 2005. PMID:16243784 doi:http://dx.doi.org/33/18/5936
- ↑ Fromme JC, Verdine GL. DNA lesion recognition by the bacterial repair enzyme MutM. J Biol Chem. 2003 Dec 19;278(51):51543-8. Epub 2003 Oct 1. PMID:14525999 doi:10.1074/jbc.M307768200
- ↑ Sugahara M, Mikawa T, Kumasaka T, Yamamoto M, Kato R, Fukuyama K, Inoue Y, Kuramitsu S. Crystal structure of a repair enzyme of oxidatively damaged DNA, MutM (Fpg), from an extreme thermophile, Thermus thermophilus HB8. EMBO J. 2000 Aug 1;19(15):3857-69. PMID:10921868 doi:http://dx.doi.org/10.1093/emboj/19.15.3857
- ↑ Gilboa R, Zharkov DO, Golan G, Fernandes AS, Gerchman SE, Matz E, Kycia JH, Grollman AP, Shoham G. Structure of formamidopyrimidine-DNA glycosylase covalently complexed to DNA. J Biol Chem. 2002 May 31;277(22):19811-6. Epub 2002 Mar 23. PMID:11912217 doi:http://dx.doi.org/10.1074/jbc.M202058200
- ↑ Zharkov DO, Golan G, Gilboa R, Fernandes AS, Gerchman SE, Kycia JH, Rieger RA, Grollman AP, Shoham G. Structural analysis of an Escherichia coli endonuclease VIII covalent reaction intermediate. EMBO J. 2002 Feb 15;21(4):789-800. PMID:11847126 doi:10.1093/emboj/21.4.789
- ↑ Imamura K, Wallace SS, Doublie S. Structural characterization of a viral NEIL1 ortholog unliganded and bound to abasic site-containing DNA. J Biol Chem. 2009 Sep 18;284(38):26174-83. Epub 2009 Jul 22. PMID:19625256 doi:10.1074/jbc.M109.021907
- ↑ Bandaru V, Zhao X, Newton MR, Burrows CJ, Wallace SS. Human endonuclease VIII-like (NEIL) proteins in the giant DNA Mimivirus. DNA Repair (Amst). 2007 Nov;6(11):1629-41. Epub 2007 Jul 12. PMID:17627905 doi:10.1016/j.dnarep.2007.05.011
- ↑ Coste F, Ober M, Carell T, Boiteux S, Zelwer C, Castaing B. Structural basis for the recognition of the FapydG lesion (2,6-diamino-4-hydroxy-5-formamidopyrimidine) by formamidopyrimidine-DNA glycosylase. J Biol Chem. 2004 Oct 15;279(42):44074-83. Epub 2004 Jul 10. PMID:15249553 doi:10.1074/jbc.M405928200
- ↑ Doublie S, Bandaru V, Bond JP, Wallace SS. The crystal structure of human endonuclease VIII-like 1 (NEIL1) reveals a zincless finger motif required for glycosylase activity. Proc Natl Acad Sci U S A. 2004 Jul 13;101(28):10284-9. Epub 2004 Jul 1. PMID:15232006 doi:10.1073/pnas.0402051101
