Structural and mechanistic studies of translesion DNA synthesis

National Institute Of Diabetes And Digestive And Kidney Diseases

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Abstract

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. Since 2001, we have studied transleson DNA synthesis (TLS) by 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. Since 2005, 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. We have studied human A-family DNA polymerase theta and human B-family DNA polymerase zeta (>500 kDa in size), which differ from the majority members in these families and carry out DNA end joining and translation synthesis. Because of the size of these polymerase-DNA complexes, we are actively using cryoEM approach. In recent years our focus is on DNA replisomes. We have assembled the simplest replisome of bacteriophage T7 DNA helicase, primes and polymerase on a DNA fork substrate and determined the first DNA replisome structure by cryoEM. Dr. Qiong Guo has revitalized the B-family translation DNA polymerase project. She has also made progresses on reconstructing E. coli DNA replication forks. With Dr. Wenqi Sun joining our group in 2023, we are making endogenously tagged replisome proteins and plan to isolate the whole replisome from cells. Dr. Wenqi Sun joint the DNA replication study in 2023 and has been focused on tagging and enriching capturing endogenously assembled replisomes. Because of the departure of Dr. Guo in 2025, Dr. Sun also inherited the B-family translesion DNA polymerase project. During 2024-2025, Dr. Young Sam Lee from DGIST, South Korea returned to NIH and helped us on the Polymerase zeta project and sped up Dr. Guo's structural characterization of human Pol zeta. We are very close to capture how Pol zeta facilitate replicative DNA polymerases to complete copying of the whole genome.