DNA is susceptible to a variety of mutations and chemical modifications. Exposure to UV irradiation, chemical agents or oxidative reagents can lead to covalent modification of DNA bases. Although most of DNA lesions are repaired by a large number of dedicated repair processes, a small number of damaged DNA sites are still encountered by DNA polymerases during genomic replication. To maintain genomic integrity and to sustain life, bacteria, archaea and eukarya use conserved mechanisms to repair and tolerate various DNA damages. My research group has continued to carry on structural and functional studies of E. coli and human translesion DNA synthesis. In the past 8 years, we have focused our studies of transleson DNA synthesis (TLS) on the Y-family DNA polymerases, which differ from normal replicative DNA polymerase in structure and function. These polymerases perform low-fidelity synthesis on undamaged DNA templates but are able to traverse normally replication-blocking lesions, including abasic sites, 8-oxo-G, benzopyrene adducts, and cyclobutane pyrimidine dimers. Y-family polymerases are widespread and enable species from E. coli to human to tolerate UV irradiation and various forms of base modification. After publishing a series of crystal structures of archaeal Y-family polymerase Dpo4 complexed with normal DNA, a cyclobutane pyrimidine dimer, benzoapyrene adduct, abasic lesion and incorrect incoming nucleotide, we have come to a conclusion that (1) the polymerase has an open and preformed active site to accommodate a variety of lesion DNA and mismatched base pairs, (2) the enzyme is specialized in bypassing abasic lesion and not other lesions and has respectable fidelity because binding of DNA lesions and incoming nucleotide does not necessarily leads to polymerization. Catalysis of the chemical step requires accurate positioning of two Mg2+ ions, and only a preferred substrate allows the Mg2+ binding in the correct geometry for chemistry. Our structural and biochemical studies suggest that both replicative and translesion DNA polymerase depend on precise metal-ion coordination for the rate-limiting step the chemical bond formation. In the last couple of years, we have expanded our TLS studies to include B-family DNA polymerases. Although many B-family DNA polymerases are involved in high fidelity genomic replication, almost every species encodes a B-family polymerase specialized in translesion DNA synthesis. Interestingly, these functionally different polymerases share many conserved sequence and structural motifs. To determine unique features that transform a replicative polymerase to carry out TLS, we have obtained crystal structures of a TLS B-family polymerase complexed with normal as well as lesion-containing DNAs. Based on the crystal structures, we have launched extensive biochemical and mutagenesis studies to probe key elements that differentiate this family of DNA polymerases to be specialized in replication or repair. The biochemical analysis together with the crystal structure reveals a novel mechanism for B family polymerase to catalyze TLS. In the current year, our focus has been placed on human B-family DNA polymerase zeta (>500 kDa in size) that differs from the majority members in the families and carries out DNA translation synthesis. Because of the size of molecule, we are actively using cryoEM approach. Another of our focus is on replication initiation. We have assembled the simplest form of DNA helicase, primes and polymerase with DNA substrate and obtained 7 initial structure using cryoEM and are in the process to get higher resolution structures.

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U.S. National Inst Diabetes/Digst/Kidney
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He, Peng; Yang, Wei (2018) Template and primer requirements for DNA Pol ?-mediated end joining. Proc Natl Acad Sci U S A 115:7747-7752
Samara, Nadine L; Gao, Yang; Wu, Jinjun et al. (2017) Detection of Reaction Intermediates in Mg2+-Dependent DNA Synthesis and RNA Degradation by Time-Resolved X-Ray Crystallography. Methods Enzymol 592:283-327
Gao, Yang; Yang, Wei (2016) Capture of a third Mg²? is essential for catalyzing DNA synthesis. Science 352:1334-7
Lee, Young-Sam; Gao, Yang; Yang, Wei (2015) How a homolog of high-fidelity replicases conducts mutagenic DNA synthesis. Nat Struct Mol Biol 22:298-303
Lee, Young-Sam; Gregory, Mark T; Yang, Wei (2014) Human Pol ? purified with accessory subunits is active in translesion DNA synthesis and complements Pol ? in cisplatin bypass. Proc Natl Acad Sci U S A 111:2954-9
Gregory, Mark T; Park, Ga Young; Johnstone, Timothy C et al. (2014) Structural and mechanistic studies of polymerase ? bypass of phenanthriplatin DNA damage. Proc Natl Acad Sci U S A 111:9133-8
Zhao, Ye; Gregory, Mark T; Biertümpfel, Christian et al. (2013) Mechanism of somatic hypermutation at the WA motif by human DNA polymerase ?. Proc Natl Acad Sci U S A 110:8146-51
Nakamura, Teruya; Zhao, Ye; Yamagata, Yuriko et al. (2012) Watching DNA polymerase ? make a phosphodiester bond. Nature 487:196-201
Kuban, Wojciech; Vaisman, Alexandra; McDonald, John P et al. (2012) Escherichia coli UmuC active site mutants: effects on translesion DNA synthesis, mutagenesis and cell survival. DNA Repair (Amst) 11:726-32
Vaisman, Alexandra; Kuban, Wojciech; McDonald, John P et al. (2012) Critical amino acids in Escherichia coli UmuC responsible for sugar discrimination and base-substitution fidelity. Nucleic Acids Res 40:6144-57

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