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Steady-state, Pre-steady-state, and Single-turnover Kinetic Measurement for DNA Glycosylase Activity

Human 8-oxoguanine DNA glycosylase (OGG1) excises the mutagenic oxidative DNA lesion 8-oxo-7,8-dihydroguanine (8-oxoG) from DNA. Kinetic characterization ...

NIEHS, National Institutes of Health

Human DNA is continuously damaged by exogenous and endogenous genotoxic insults. To counteract DNA damage and ensure the completion of DNA replication, cells possess specialized DNA polymerases (Pols) that bypass a variety of DNA lesions. Human DNA polymerase kappa (hPolkappa) is a member of the Y-family of DNA Pols and a direct counterpart of DinB in Escherichia coli. hPolkappa is characterized by its ability to bypass several DNA adducts [e.g., benzo[a]pyrene diolepoxide-N(2)-deoxyguanine (BPDE-N(2)-dG) and thymine glycol] and efficiently extend primers with mismatches at the termini. hPolkappa is structurally distinct from E. coli DinB in that it possesses an approximately 100-amino acid extension at the N-terminus. Here, we report that tyrosine 112 (Y112), the steric gate amino acid of hPolkappa, which distinguishes dNTPs from rNTPs by sensing the 2'-hydroxy group of incoming nucleotides, plays a crucial role in extension reactions with mismatched primer termini. When Y112 was replaced with alanine, the amino acid change severely reduced the catalytic constant, i.e., k(cat), of the extending mismatched primers and lowered the efficiency, i.e., k(cat)/K(m), of this process by approximately 400-fold compared with that of the wild-type enzyme. In contrast, the amino acid replacement did not reduce the insertion efficiency of dCMP opposite BPDE-N(2)-dG in template DNA, nor did it affect the ability of hPolkappa to bind strongly to template-primer DNA with BPDE-N(2)-dG/dCMP. We conclude that the steric gate of hPolkappa is a major fidelity factor that regulates extension reactions from mismatched primer termini.

The steric gate amino acid tyrosine 112 is required for efficient mismatched-primer extension by human DNA polymerase kappa.

Oxidized DNA precursors can cause mutagenesis and carcinogenesis when they are incorporated into the genome. Some human Y-family DNA polymerases (Pols) can effectively incorporate 8-oxo-dGTP, an oxidized form of dGTP, into a position opposite a template dA. This inappropriate G:A pairing may lead to transversions of A to C. To gain insight into the mechanisms underlying erroneous nucleotide incorporation, we changed amino acids in human Poleta and Polkappa proteins that might modulate their specificity for incorporating 8-oxo-dGTP into DNA. We found that Arg61 in Poleta was crucial for erroneous nucleotide incorporation. When Arg61 was substituted with lysine (R61K), the ratio of pairing of dA to 8-oxo-dGTP compared to pairing of dC was reduced from 660:1 (wild-type Poleta) to 7 : 1 (R61K). Similarly, Tyr112 in Polkappa was crucial for erroneous nucleotide incorporation. When Tyr112 was substituted with alanine (Y112A), the ratio of pairing was reduced from 11: 1 (wild-type Polkappa) to almost 1: 1 (Y112A). Interestingly, substitution at the corresponding position in Poleta, i.e. Phe18 to alanine, did not alter the specificity. These results suggested that amino acids at distinct positions in the active sites of Poleta and Polkappa might enhance 8-oxo-dGTP to favor the syn conformation, and thus direct its misincorporation into DNA.

Critical amino acids in human DNA polymerases eta and kappa involved in erroneous incorporation of oxidized nucleotides.

Human cells possess multiple specialized DNA polymerases (Pols) that bypass a variety of DNA lesions which otherwise would block chromosome replication. Human polymerase kappa (Pol Îº) bypasses benzo[a]pyrene diolepoxide-N(2)-deoxyguanine (BPDE-N(2)-dG) DNA adducts in an almost error-free manner. To better understand the relationship between the structural features in the active site and lesion bypass by Pol Îº, we mutated codons corresponding to amino acids appearing close to the adducts in the active site, and compared bypass efficiencies. Remarkably, the substitution of alanine for phenylalanine 171 (F171), an amino acid conserved between Pol Îº and its bacterial counterpart Escherichia coli DinB, enhanced the efficiencies of dCMP incorporation opposite (-)- and (+)-trans-anti-BPDE-N(2)-dG 18-fold. This substitution affected neither the fidelity of TLS nor the efficiency of dCMP incorporation opposite normal guanine. This amino acid change also enhanced the binding affinity of Pol Îº to template/primer DNA containing (-)-trans-anti-BPDE-N(2)-dG. These results suggest that F171 functions as a molecular brake for TLS across BPDE-N(2)-dG by Pol Îº and that the F171A derivative of Pol Îº bypasses these DNA lesions more actively than does the wild-type enzyme.

Phenylalanine 171 is a molecular brake for translesion synthesis across benzo[a]pyrene-guanine adducts by human DNA polymerase kappa.

Chronic inflammation is known to lead to an increased risk for the development of cancer. Under inflammatory condition, cellular DNA is damaged by hypobromous acid, which is generated by myeloperoxidase and eosinophil peroxidase. The reactive brominating species induced brominated DNA adducts such as 8-bromo-2'-deoxyguanosine (8-Br-dG), 8-bromo-2'-deoxyadenosine (8-Br-dA), and 5-bromo-2'-deoxycytidine (5-Br-dC). These DNA lesions may be implicated in carcinogenesis. In this study, we analyzed the miscoding properties of the brominated DNA adducts generated by human DNA polymerases (pols). Site-specifically modified oligodeoxynucleotides containing a single 8-Br-dG, 8-Br-dA, or 5-Br-dC were used as a template in primer extension reactions catalyzed by human pols Î±, Îº, and Î·. When 8-Br-dG-modified template was used, pol Î± primarily incorporated dCMP, the correct base, opposite the lesion, along with a small amount of one-base deletion (4.8%). Pol Îº also promoted one-base deletion (14.2%), accompanied by misincorporation of dGMP (9.5%), dAMP (8.0%), and dTMP (6.1%) opposite the lesion. Pol Î·, on the other hand, readily bypassed the 8-Br-dG lesion in an error-free manner. As for 8-Br-dA and 5-Br-dC, all the pols bypassed the lesions and no miscoding events were observed. These results indicate that only 8-Br-dG, and not 5-Br-dC and 8-Br-dA, is a mutagenic lesion; the miscoding frequency and specificity vary depending on the DNA pol used. Thus, hypobromous acid-induced 8-Br-dG adduct may increase mutagenic potential at the site of inflammation.

Mutational specificities of brominated DNA adducts catalyzed by human DNA polymerases.

Human 8-oxoguanine DNA glycosylase (OGG1) is a key enzyme involved in removing 7,8-dihydro-8-oxoguanine (8-oxoG), a highly mutagenic DNA lesion generated by oxidative stress. The removal of 8-oxoG by OGG1 is affected by the local DNA sequence, and this feature most likely contributes to observed mutational hot spots in genomic DNA. To elucidate the influence of local DNA sequence on 8-oxoG excision activity of OGG1, we conducted steady-state, pre-steady-state, and single turnover kinetic evaluation of OGG1 in alternate DNA sequence contexts. The sequence context effect was studied for a mutational hot spot at a CpG dinucleotide. Altering either the global DNA sequence or the 5'-flanking unmodified base pair failed to influence the excision of 8-oxoG. Methylation of the cytosine 5' to 8-oxoG also did not affect 8-oxoG excision. In contrast, a 5'-neighboring mismatch strongly decreased the rate of 8-oxoG base removal. Substituting the 5'-C in the CpG dinucleotide with T, A, or tetrahydrofuran (i.e. T:G, A:G, and tetrahydrofuran:G mispairs) resulted in a 10-, 13-, and 4-fold decrease in the rate constant for 8-oxoG excision, respectively. A greater loss in activity was observed when T:C or A:C was positioned 5' of 8-oxoG (59- and 108-fold, respectively). These results indicate that neighboring structural abnormalities 5' to 8-oxoG deter its repair thereby enhancing its mutagenic potential.

DNA sequence context effects on the glycosylase activity of human 8-oxoguanine DNA glycosylase.

Many chronic inflammatory conditions are associated with an increased risk of cancer development. At the site of inflammation, cellular DNA is damaged by hypochlorous acid (HOCl), a potent oxidant generated by myeloperoxidase. 8-Chloro-2'-deoxyguanosine (8-Cl-dG) is a major DNA adduct formed by HOCl and has been detected from the liver DNA and urine of rats administered lipopolysaccharide in an inflammation model. Thus, the 8-Cl-dG lesion may be associated with the carcinogenesis of inflamed tissues. In this study, we explored the miscoding properties of the 8-Cl-dG adduct generated by human DNA polymerases (pols). Site-specifically modified oligodeoxynucleotide containing a single 8-Cl-dG was prepared and used as a template in primer extension reactions catalysed by human pol Î±, Ä¸ or Î·. Primer extension reactions catalysed by pol Î± and Ä¸ in the presence of all four dNTPs were slightly retarded at the 8-Cl-dG site, while pol Î· readily bypassed the lesion. The fully extended products were analysed to quantify the miscoding frequency and specificity of 8-Cl-dG using two-phased polyacrylamide gel electrophoresis (PAGE). During the primer extension reaction in the presence of four dNTPs, pol Ä¸ promoted one-base deletion (6.4%), accompanied by the misincorporation of 2'-deoxyguanosine monophosphate (5.5%), dAMP (3.7%), and dTMP (3.5%) opposite the lesion. Pol Î± and Î·, on the other hand, exclusively incorporated dCMP opposite the lesion. The steady-state kinetic studies supported the results obtained from the two-phased PAGE assay. These results indicate that 8-Cl-dG is a mutagenic lesion; the miscoding frequency and specificity varies depending on the DNA polymerase used. Thus, HOCl-induced 8-Cl-dG adduct may be involved in inflammation-driven carcinogenesis.

Akira Sassa

Laboratory of Structural Biology

NIEHS, National Institutes of Health

Steady-state, Pre-steady-state, and Single-turnover Kinetic Measurement for DNA Glycosylase Activity

Akira Sassa

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Laboratory of Structural Biology

NIEHS, National Institutes of Health

Steady-state, Pre-steady-state, and Single-turnover Kinetic Measurement for DNA Glycosylase Activity

Laboratory of Structural Biology