DNA Mismatch Repair by MutH
DNA Mismatch Repair (MMR) occurs when a mismatch of DNA bases occurs during DNA replication that is not corrected by the polymerases. This mismatch can be at a single nucleotide or an insertion or deletion of up to 4 bases. An integral protein in MMR is MutH. MutH is an endonuclease, which means it is an enzyme that can digest DNA in the middle of the sequence. However, it is a weak endonuclease so it will only cause a single-stranded nick upstream or downstream of the damaged daughter strand DNA and not the correct parent strand. This allows it to be re-replicated as the correct sequence by DNA polymerase. Homodimers of MutS and MutL bind the mismatched DNA and create a loop that MutH can bind to. Therefore, MutS and MutL are necessary to recruit MutH to nick the DNA. In order to maintain the correct DNA sequence and repair the damaged portion without mutations, MutH must be able to differentiate the incorrect daughter strand from the correct parent strand. In bacteria, the freshly replicated DNA is hemimethylated, meaning that the parent strand is methylated and the daughter strand has not yet been methylated by methyltransferases. MutH then nicks the phosphodiester bond 5' of a GATC palindrome on the umethylated daughter strand. The GATC palindrome can be upstream or downstream of the damaged DNA site by up to 1000 nucleotides. This allows the damaged strand to be destroyed by exonucleases and re-replicated by DNA polymerase as the correct sequence.
Structure of MutH
MutH has two subdomains, the "N" arm and the "C"arm which is based on the N and C termini of the protein. These arms are arranged in a . The N arm contains the catalytic core consisting of the and an essential Glu56 residue. The catalytic core is where the endonuclease reaction of hydrolyzing the phosphodiester bond occurs. The DEK motif consists of Asp(D)-X(n)-Glu(E)-X-Lys(K) sequence, which contains the Mg2+ required for nicking the phosphodiester bond. The DEK motif is found in most endonucleases, which highlights its importance in catalyzing the hydrolysis of the phosphodiester bond.
The is responsible for base recognition and sequence-specific binding of the DNA. The cleft in the V binds the DNA. The C-term residues help to bind the N-arm and are shown to increase DNA binding in the closed position. This allows it to have the correct shape and chemical interactions to bind the damaged daughter strand DNA substrate and catalyze the hydrolysis reaction in the correct location.
MutH must be able to correctly recognize the GATC palindrome of the damaged umethylated daughter strand in order to cleave it properly. The secondary structure of Beta sheets 3/9/6 and loop 67 of arm "C" bind the GATC sequence in the major groove of the DNA. The N-arm contacts 6 nucleotides of the cleavage strand in the minor groove of the DNA. Lys45/Asp46 interacts with the phosphate backbone to narrow the minor groove of the DNA. Loop C1 Ser65 H-bonds the nitrogen of Ala67 to stabilize the loop. (residues 184-190) binds the GATC motif. The G and C are hydrogen bonded by residues Asp184/Glu91 and Lys186/Gly187. Tyr212 bonds N6 the of unmodified adenine and Pro185 interacts with methylated adenine. These specific bonds allow for the recognition of hemimethylated DNA and differentiate the parent strand from the daughter strand. Loop BC Lys48 binds the oxygens of the T’s. The active (catalytic) site on the N arm is Glu56, Asp70, Glu77, and Lys79, this makes up the . The carboxylates (Glu/Asp) coordinate two Ca+ ions in the active site. Lys79 links the two arms of MutH and allows for the sequence-specific cutting of DNA. the reaction is catalyzed by Lys79, the 3’ phosphate of DNA that is upstream of the GATC palindrome, and the nearby metal ions to activate water for a to create a single-stranded nick in the daughter strand 5' to the palindrome. Once the nick is created, the damaged daughter strand can be destroyed and re-replicated correctly.
References
Ban, C., & Yang, W. (1998). Structural basis for MutH activation in E.coli mismatch repair and relationship of MutH to restriction endonucleases. The EMBO
journal, 17(5), 1526–1534. https://doi.org/10.1093/emboj/17.5.1526
Lee, J. Y., Chang, J., Joseph, N., Ghirlando, R., Rao, D. N., & Yang, W. (2005). MutH complexed with hemi- and unmethylated DNAs: coupling base recognition
and DNA cleavage. Molecular cell, 20(1), 155–166. https://doi.org/10.1016/j.molcel.2005.08.019
Voet, D., Voet, J. G., & Pratt, C. W. (2013). Fundamentals of Biochemistry: Life at the molecular level. Wiley.
RecA Protein Structure and Function
Monomer Structure
is one of the many proteins that is involved in recombination cross-over events and during recombination repair in response to single strand DNA breaks. RecA is a rather small monomer protein that can multiplex with itself up to thousands of RecA proteins to associate with ssDNA. The structure of RecA was determined through x-ray crystallography and each monomer contains very distinct structural components. These are a largely helical 30-residue N-terminal region, a 240-residue α/ß ATPase core, and a 64-residue C-terminal
globular domain.
Monomer Association
The process of recruiting new RecA monomers is carried out through an ATP-dependent process. This occurs through the binding of ATP to two adjacent on subsequent RecA monomers. To properly grow the crystal that was used to determine structure through x-ray crystallography, a non-hydrolyzable analog of ATP is used. This analog has the shorthand formula of ADP-AlF4-Mg. Specifically, the aluminum tetrafluoride is bound to the adenine diphosphate in the ɣ position. Several residues are involved in the hydrolysis of ATP to coordinate strand exchange after binding to ssDNA has occurred. On one of the RecA monomers, two lysine residues, Lys 248 and Lys 250, are responsible for coordinating with the ɣ phosphate stabilizing it. Lys 250 has also been implicated to have an additional function: to coordinate a glutamic acid, Glu 96, on the adjacent RecA monomer. This coordination with Glu 96 is achieved through hydrogen bonding and is believed to be critical for the catalytic mechanism. Specifically, there is a complex network of hydrogen bonding that is occurring between several other residues to rotate Glu 96 to a more favorable conformation enabling Glu 96 to act as a nucleophile.
ssDNA Binding
Once several RecA monomers have coordinated with one another, they coordinate with ssDNA to form a repeating structure that contains exactly three nucleotides for every RecA monomer. However, this does not mean that each nucleotide triplet only interacts with a single RecA monomer. In reality, each RecA monomer spans three nucleotides, but the nucleotide triplet interacts with the other two RecA surrounding it in both the 5' and 3' direction. Essentially, each nucleotide triplet is interacting with three different RecA monomers named RecA5', RecA0, and RecA3' based on their relative location to the nucleotide triplet. The first nucleotide of the triplet is bound by both RecA5' and RecA0, the second is bound only by Rec0 and the third is bound by both Rec0 and Rec3'. is responsible for stabilizing ssDNA within this conformation. Specifically, the phosphate backbone of the nucleotide triplet is what interacts with the RecA monomer residues through hydrogen bonding (dashed lines). Interestingly, the hydrogen bonding interactions that are occurring do not always use the side chains, but often will interact with the amide groups on amino acid backbones. For example, the first phosphate group within a nucleotide triplet will interact with the backbone amide of Met 197 from RecA5' and the amide backbone of Asn 123 from RecA0. The second phosphate of the triplet interacts with Gly 211 and Gly 212 on RecA0. The third phosphate of the triplet is unique as it interacts with the side chains of Ser 172 and Arg 176.
Strand Exchange Mechanism
Once a RecA filament has properly formed and coordinated with ssDNA, a complementary DNA strand must be located. Once a complementary strand is located, the donor is wound into the filament complex where the ssDNA and dsDNA form a temperate three-stranded DNA intermediate. Another protein complex, RecBCD, not modeled here, helps resolve the strand exchange process via the formation of a holiday junction. The process of strand exchange is heavily mediated through traditional Watson-Crick base pairing rules, but also by a few residues located within the RecA filament complex. Specifically, Ser 162 on each RecA monomer contacts the phosphate groups near the nucleotide triplet. Additionally, Met 164 increases the spacing between nucleotide triplets by inserting itself into the gap between them. This insertion allows for more strict base pairing stabilization. The final residue implicated in strand exchange appears to have a proofreading mechanism similar to that of DNA polymerases. This residue is and has been implicated in having base-pairing proofreading abilities by hydrogen bonding with O2 groups in thymidine bases (DT 7-9). This interaction is able to check for proper Watson-Crick base pairing because the bond lengths associated with proper base pairing will allow for proper interactions between Arg 169 and thymidine. Incorrect base pairing will cause thymidine residues to shift position preventing necessary Arg 169 interactions. This functionality has been shown through the mutation of Arg 169 to Histidine resulting in ultraviolet sensitivity and increased mismatched base pairing. However, this proofreading mechanism is not completely understood and this functionality may be a fragment of Arg 169 being able to interact with subsequent thymidine bases used in the crystalized DNA structure.
References
1. Chen, Z., Yang, H., & Pavletich, N. P. (2008). Mechanism of homologous recombination from the RecA-ssDNA/dsDNA structures. Nature, 453(7194), 489–494. https://doi.org/10.1038/nature06971
2.Voet, D., Voet, J. G., & Pratt, C. W. (2013). Fundamentals of biochemistry : life at the molecular level. Wiley.
3. Yang, H., Zhou, C., Dhar, A., & Pavletich, N. P. (2020). Mechanism of strand exchange from RecA–DNA synaptic and D-loop structures. Nature, 586(7831), 801–806. https://doi.org/10.1038/s41586-020-2820-9
UvrD
, also known as Helicase II, is one of many components responsible in repairing DNA damage. Helicases use energy from ATP to unwind double helices in metabolic pathways using nucleic acids. ATP molecules are typically used to store energy shared between phosphate groups that gets released when breaking bonds to drive catabolic reactions.
Helicases were found in the 1970’s to be DNA-dependent ATPases, meaning that they use ATP hydrolysis to complete its interactions with the different types of nucleic acids it comes into contact with. Helicase II, also called UvrD is the founding member of SF1, one group of six superfamiliies used to identify helicases. SF1 and SF2 members share seven conserved sequence motifs that are involved in ATP Binding [1]. UvrD is important in replication, recombination, and repair from ultraviolet damage and mismatched base pairs. Nucleotide excision repair in a normal cell is supposed to correct pyrimidine dimers and other DNA lesions when bases are displaced from their normal positions. UvrD pairs up with the UvrABC endonuclease system, which works to displace the DNA. This is then repaired by PolI and DNA ligase [2].
UvrD Motifs
There are for UvrD, which are conserved in other homologous structures. The homologous structures mentioned are Helicase 2 homologs, which appear in different species. These conserved motifs are important to maintain the function of UvrD. There are 4 domains that these motifs fit into (not shown). The domains are 1A, 1B, 2A, and 2B. Motifs I, Ia, II-VI are involved in ATP binding. Motifs Ia, III, and V are involved in ssDNA binding. Motif IV is reported to be unique in SF1. They found in their paper, seven new sequence motifs conserved among UvrD homologs. They are Ib, Ic, Id, IVb, IVc, Va, and VIa. These conserved residues are involved in DNA binding or domain 1B and 2B interactions [1].
Separation Pin
The "" is a part of the 2B domain and is responsible for unwinding the DNA. This uses a 2 step power stroke, one stroke when ATP is bound and another stroke when ADP and Pi are released. The GIG motif and separation pin work together to unwind the DNA and move it out of the way so UvrD can unwind more DNA. The separation pin also prevents ssDNA once unwound from moving backwards and from reannealing. The proposed method is called the wrench-and-inchworm method, which is when the enzyme binds DNA and attaches at different points and then moves 1 nucleotide per ATP molecule.After an ATP molecule is released, UvrD is then ready to proceed forward to the next nucleotide [1].
UvrD Binding Site for ATP analog (AMPPNP)
When determining the structure of UvrD, an ATP analog was used. They used an so that the last phosphate can't be cleaved. Using the unhydrolyzable analog is beneficial in locking in the structure to observe.The green ion shown in the ATP analog scene is a Mg2+ ion, which is essential for ATP hydrolysis and interacts with the β and γ phosphates. The magnesium ion is surrounded by essential residues that when altered, have been shown to have reduced ATPase activity [1].
UvrD Binding Site for ATP analog (ADP•MgF3)
To capture the UvrD-DNA-ADP complex, a new crystal structure used ADP•MgF3 after NaF was added to help improve crystal growth. This structure is believed to be a more authentic transition state analog, which differs from the AMPPNP analog slightly. The has a , which is a glycerol molecule, which has hydrogen bonding similar to interactions that E566 has to a 3' OH of the ribose. The DNA isn't actually bound in the crystal structure, but can be used to visualize what hydrogen bonding might look like when connected to the backbone in DNA. , which typically would bind to the 3' OH of the ribose of DNA. Another residue, R37 (Not Shown), binds to the 2' OH of ribose, which has weaker hydrogen bonding. This is a structural component that allows UvrD to bind both ATP and dATP[1].
DNA Repair Mechanism; URACIL-DNA GLYCOSYLASE
Glycosylase is an enzyme. Its main function is in Base Excision Repair(BER). Base Excision Repair is a DNA repair mechanism that fixes the most common type of DNA damage. BER corrects DNA damage that occurs from oxidation and methylation. BER removes and repairs damaged bases usually these are single-stranded DNA breaks. It also corrects DNA damage that results from small leisures that do not disrupt the double helix[3].
Function
Glycosylase does this by cleaving the glycosidic bond of the damaged nucleotide, leaving the Deoxyribose nucleotide with no base. The deoxyribose is then cleaved by AP endonuclease creating an AP site. The gap that is left is filled in through DNA Polymerase and DNA ligase[4].
Uracil-DNA Glycosylase
The structure of Glycosylase has a couple of different forms in terms of its general structure there is Adenine and Uracil Glycosylase. DNA Uracil-Glycosylase specifically looks for any Uracil in the double-stranded DNA. It looks for Uracil in dsDNA because uracil is only found in RNA. So if a Uracil is found in dsDNA then that means one of the strands has been damaged and needs repair. The dsDNA in the 3D model contains a U G base pair mismatch. When Uracil-DNA Glycosylase finds the site it binds to it. Then a nucleotide-flipping mechanism flips the site of repair out of the double helix. The of Uracil Glycosylase; D145, Y147, F158, N204, H268, L272 is what binds to the double-stranded DNA with the damaged lesion. This is what allows the and flipping of the damaged site out of the double helix. ASN 204 and HIS 268 are responsible for catalyzing the cleavage of the glycosidic bond. TYR 147, PHE 158, and ASN 204 all aid in Uracil excision and replacement with Thymine. When flipped the damaged bases out of the helix takes its place in the minor groove since AP sites can be mutagenic[5]. The Uracil is then replaced with a Thymine. This is because Uracil and Thymine have identical base pairing properties. Thymine happens to have greater resistance to photochemical mutations which is why we see it in dsDNA and not Uracil.
