Polymerase manager protein UmuD directly regulates Escherichia coli DNA polymerase III α binding to ssDNA.

Replication by Escherichia coli DNA polymerase III is disrupted on encountering DNA damage. Consequently, specialized Y-family DNA polymerases are used to bypass DNA damage. The protein UmuD is extensively involved in modulating cellular responses to DNA damage and may play a role in DNA polymerase exchange for damage tolerance. In the absence of DNA, UmuD interacts with the α subunit of DNA polymerase III at two distinct binding sites, one of which is adjacent to the single-stranded DNA-binding site of α. Here, we use single molecule DNA stretching experiments to demonstrate that UmuD specifically inhibits binding of α to ssDNA. We predict using molecular modeling that UmuD residues D91 and G92 are involved in this interaction and demonstrate that mutation of these residues disrupts the interaction. Our results suggest that competition between UmuD and ssDNA for α binding is a new mechanism for polymerase exchange.

Structural basis for cisplatin DNA damage tolerance by human polymerase η during cancer chemotherapy.

A major clinical problem in the use of cisplatin to treat cancers is tumor resistance. DNA polymerase η (Pol-η) is a crucial polymerase that allows cancer cells to cope with the cisplatin-DNA adducts that are formed during chemotherapy. We present here a structure of human Pol-η inserting deoxycytidine triphosphate (dCTP) opposite a cisplatin intrastrand cross-link (PtGpG). We show that the specificity of human Pol-η for PtGpG derives from an active site that is open to permit Watson-Crick geometry of the nascent PtGpG-dCTP base pair and to accommodate the lesion without steric hindrance. This specificity is augmented by the residues Gln38 and Ser62, which interact with PtGpG, and Arg61, which interacts with the incoming dCTP. Collectively, the structure provides a basis for understanding how Pol-η in human cells can tolerate the DNA damage caused by cisplatin chemotherapy and offers a framework for the design of inhibitors in cancer therapy.

Role of human DNA polymerase κ in extension opposite from a cis-syn thymine dimer.

Exposure of DNA to UV radiation causes covalent linkages between adjacent pyrimidines. The most common lesion found in DNA from these UV-induced linkages is the cis-syn cyclobutane pyrimidine dimer. Human DNA polymerase κ (Polκ), a member of the Y-family of DNA polymerases, is unable to insert nucleotides opposite the 3'T of a cis-syn T-T dimer, but it can efficiently extend from a nucleotide inserted opposite the 3'T of the dimer by another DNA polymerase. We present here the structure of human Polκ in the act of inserting a nucleotide opposite the 5'T of the cis-syn T-T dimer. The structure reveals a constrained active-site cleft that is unable to accommodate the 3'T of a cis-syn T-T dimer but is remarkably well adapted to accommodate the 5'T via Watson-Crick base pairing, in accord with a proposed role for Polκ in the extension reaction opposite from cyclobutane pyrimidine dimers in vivo.

Structure of human DNA polymerase kappa inserting dATP opposite an 8-OxoG DNA lesion.

BACKGROUND: Oxygen-free radicals formed during normal aerobic cellular metabolism attack bases in DNA and 7,8-dihydro-8-oxoguanine (8-oxoG) is one of the major lesions formed. It is amongst the most mutagenic lesions in cells because of its dual coding potential, wherein 8-oxoG(syn) can pair with an A in addition to normal base pairing of 8-oxoG(anti) with a C. Human DNA polymerase kappa (Polkappa) is a member of the newly discovered Y-family of DNA polymerases that possess the ability to replicate through DNA lesions. To understand the basis of Polkappa's preference for insertion of an A opposite 8-oxoG lesion, we have solved the structure of Polkappa in ternary complex with a template-primer presenting 8-oxoG in the active site and with dATP as the incoming nucleotide. METHODOLOGY AND PRINCIPAL FINDINGS: We show that the Polkappa active site is well-adapted to accommodate 8-oxoG in the syn conformation. That is, the polymerase and the bound template-primer are almost identical in their conformations to that in the ternary complex with undamaged DNA. There is no steric hindrance to accommodating 8-oxoG in the syn conformation for Hoogsteen base-paring with incoming dATP. CONCLUSIONS AND SIGNIFICANCE: The structure we present here is the first for a eukaryotic translesion synthesis (TLS) DNA polymerase with an 8-oxoG:A base pair in the active site. The structure shows why Polkappa is more efficient at inserting an A opposite the 8-oxoG lesion than a C. The structure also provides a basis for why Polkappa is more efficient at inserting an A opposite the lesion than other Y-family DNA polymerases.

Human DNA polymerase kappa encircles DNA: implications for mismatch extension and lesion bypass.

Human DNA polymerase kappa (Pol kappa) is a proficient extender of mispaired primer termini on undamaged DNAs and is implicated in the extension step of lesion bypass. We present here the structure of Pol kappa catalytic core in ternary complex with DNA and an incoming nucleotide. The structure reveals encirclement of the DNA by a unique "N-clasp" at the N terminus of Pol kappa, which augments the conventional right-handed grip on the DNA by the palm, fingers, and thumb domains and the PAD and provides additional thermodynamic stability. The structure also reveals an active-site cleft that is constrained by the close apposition of the N-clasp and the fingers domain, and therefore can accommodate only a single Watson-Crick base pair. Together, DNA encirclement and other structural features help explain Pol kappa's ability to extend mismatches and to promote replication through various minor groove DNA lesions, by extending from the nucleotide incorporated opposite the lesion by another polymerase.

The role of specific amino acid residues in the active site of Escherichia coli DNA polymerase I on translesion DNA synthesis across from and past an N-2-aminofluorene adduct.

Understanding how carcinogenic DNA adducts compromise accurate DNA replication is an important goal in cancer research. A central part of these studies is to determine the molecular mechanism that allows a DNA polymerase to incorporate a nucleotide across from and past a bulky adduct in a DNA template. To address the importance of polymerase architecture on replication across from this type of bulky DNA adduct, three active-site mutants of Escherichia coli DNA polymerase I (Klenow fragment) were used to study DNA synthesis on DNA modified with the carcinogen N-2-aminofluorene (AF). Running-start synthesis studies showed that full-length synthesis past the AF adduct was inhibited for all of the mutants, but that this inhibition was substantially less for the F762A mutant. Single nucleotide extension and steady-state kinetic experiments showed that the Y766S mutant displayed higher rates of insertion of each incorrect nucleotide relative to WT across from the dG-AF adduct. This effect was not observed for F762A or E710A mutants. Similar experiments that measured synthesis one nucleotide past the dG-AF adduct revealed an enhanced preference by the F762A mutant for dG opposite the T at this position. Finally, synthesis at the +1 and +2 positions was inhibited to a greater extent for the Y766S and E710A mutants compared with both the WT and F762A mutants. Taken together, this work is consistent with the model that polymerase geometry plays a crucial role in both the insertion and extension steps during replication across from bulky DNA lesions.

Mechanistic insights into replication across from bulky DNA adducts: a mutant polymerase I allows an N-acetyl-2-aminofluorene adduct to be accommodated during DNA synthesis.

The molecular mechanism that allows a polymerase to incorporate a nucleotide opposite a DNA lesion is not well-understood. One way to study this process is to characterize the altered molecular interactions that occur between the polymerase and a damaged template. Prior studies have determined the polymerase-template dissociation constants and used kinetic analyses and a protease digestion assay to measure the effect of various DNA adducts positioned in the active site of Klenow fragment (KF). Here, a mutator polymerase was used in which the tyrosine at position 766 of the KF has been replaced with a serine. This position is located at the junction of the fingers and palm domain and is thought to be involved in maintaining the active site geometry. The primer-template was modified with N-acetyl-2-aminofluorene (AAF), a well-studied carcinogenic adduct. The mutant polymerase displayed a significant increase in the rate of incorporation of the correct nucleotide opposite the adduct but was much less prone to incorporate an incorrect nucleotide relative to the wild-type polymerase. Both the wild-type and the mutant polymerase bound much more tightly to the AAF-modified primer-template; however, unlike the wild-type polymerase, the binding strength of the mutant was influenced by the presence of a dNTP. Moreover, the mutant polymerase was able to undergo a dNTP-induced conformational change when the AAF adduct was positioned in the active site, while the wild-type enzyme could not. A model is proposed in which the looser active site of the mutant is able to better accommodate the AAF adduct.