Structural highlights
Function
[DPOLB_HUMAN] Repair polymerase that plays a key role in base-excision repair. Has 5'-deoxyribose-5-phosphate lyase (dRP lyase) activity that removes the 5' sugar phosphate and also acts as a DNA polymerase that adds one nucleotide to the 3' end of the arising single-nucleotide gap. Conducts 'gap-filling' DNA synthesis in a stepwise distributive fashion rather than in a processive fashion as for other DNA polymerases.[1] [2] [3] [4]
Publication Abstract from PubMed
A wide variety of endogenous and exogenous alkylating agents attack DNA to preferentially generate N7-alkylguanine (N7-alkylG) adducts. Studies on the effect of N7-alkylG lesions on biological processes have been difficult due in part to complications arising from the chemical lability of the positively charged N7-alkylG, which can readily produce secondary lesions. To assess the effect of bulky N7-alkylG on DNA replication, we prepared chemically stable N7-benzylguanine(N7bnG)-containing DNA and evaluated nucleotide incorporation opposite the lesion by human DNA polymerase beta (polbeta), a model enzyme for high-fidelity DNA polymerases. Kinetic studies showed that the N7-benzyl-G lesion greatly inhibited dCTP incorporation by polbeta. The crystal structure of polbeta incorporating dCTP opposite N7bnG showed a Watson-Crick N7bnG:dCTP. The polbeta-N7bnG:dCTP structure showed an open protein conformation, a relatively disordered dCTP, and lack of catalytic metal, which explained the inefficient nucleotide incorporation opposite N7bnG. This indicates that polbeta is sensitive to major groove adducts in the templating-base side and deters nucleotide incorporation opposite bulky N7-alkylG adducts by adopting a catalytically incompetent conformation. Substituting Mg2+ for Mn2+ induced an open-to-closed conformational change due to the presence of catalytic metal and stably bound dCTP and increased the catalytic efficiency by ~10-fold, highlighting the effect of binding of incoming nucleotide and catalytic metal on protein not conformation and nucleotidyl transfer reaction. Overall, these results suggest that, although bulky alkyl groups at guanine-N7 may not alter base-pairing properties of guanine, the major-groove-positioned lesions in the template could impede nucleotidyl transfer by some DNA polymerases.
Structural and kinetic studies of the effect of guanine-N7 alkylation and metal cofactors on DNA replication.,Kou Y, Koag MC, Lee S Biochemistry. 2018 Jun 29. doi: 10.1021/acs.biochem.8b00331. PMID:29957995[5]
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.
See Also
References
- ↑ Bennett RA, Wilson DM 3rd, Wong D, Demple B. Interaction of human apurinic endonuclease and DNA polymerase beta in the base excision repair pathway. Proc Natl Acad Sci U S A. 1997 Jul 8;94(14):7166-9. PMID:9207062
- ↑ Matsumoto Y, Kim K, Katz DS, Feng JA. Catalytic center of DNA polymerase beta for excision of deoxyribose phosphate groups. Biochemistry. 1998 May 5;37(18):6456-64. PMID:9572863 doi:10.1021/bi9727545
- ↑ DeMott MS, Beyret E, Wong D, Bales BC, Hwang JT, Greenberg MM, Demple B. Covalent trapping of human DNA polymerase beta by the oxidative DNA lesion 2-deoxyribonolactone. J Biol Chem. 2002 Mar 8;277(10):7637-40. Epub 2002 Jan 22. PMID:11805079 doi:10.1074/jbc.C100577200
- ↑ Parsons JL, Dianova II, Khoronenkova SV, Edelmann MJ, Kessler BM, Dianov GL. USP47 is a deubiquitylating enzyme that regulates base excision repair by controlling steady-state levels of DNA polymerase beta. Mol Cell. 2011 Mar 4;41(5):609-15. doi: 10.1016/j.molcel.2011.02.016. PMID:21362556 doi:10.1016/j.molcel.2011.02.016
- ↑ Kou Y, Koag MC, Lee S. Structural and kinetic studies of the effect of guanine-N7 alkylation and metal cofactors on DNA replication. Biochemistry. 2018 Jun 29. doi: 10.1021/acs.biochem.8b00331. PMID:29957995 doi:http://dx.doi.org/10.1021/acs.biochem.8b00331
