Pol β lyase removal of 5′-dRP from BER intermediates is critical in mammalian BER involving the abasic site. We found that pol β also removes 5′-adenylated dRP from BER intermediates after abortive ligation. The crystal structure of a pol β-DNA complex revealed that the 5′-AMP-dRP group is positioned in the lyase active site. Expression of pol β rescued MMS sensitivity in APTX (hnt3) and FEN1 (rad27) deficient yeast.

Background: Human 8-oxoguanine DNA glycosylase (OGG1) removes the mutagenic DNA lesion 7,8-dihydro-8-oxoguanine. Results: OGG1 activity is significantly decreased by an adjacent DNA mismatch. Conclusion: Tandem lesions can dramatically diminish base excision repair. Significance: Deamination of 5Ј-methylcytosine in CpG-rich DNA sequences prone to oxidative DNA damage could be a significant factor in the generation of G to T transversions hot spots.

Edited by Patrick SungNumerous ribonucleotides are incorporated into the genome during DNA replication. Oxidized ribonucleotides can also be erroneously incorporated into DNA. Embedded ribonucleotides destabilize the structure of DNA and retard DNA synthesis by DNA polymerases (pols), leading to genomic instability. Mammalian cells possess translesion DNA synthesis (TLS) pols that bypass DNA damage. The mechanism of TLS and repair of oxidized ribonucleotides remains to be elucidated. To address this, we analyzed the miscoding properties of the ribonucleotides riboguanosine (rG) and 7,8-dihydro-8-oxo-riboguanosine (8-oxo-rG) during TLS catalyzed by the human TLS pols and in vitro. The primer extension reaction catalyzed by human replicative pol ␣ was strongly blocked by 8-oxo-rG. pol inefficiently bypassed rG and 8-oxo-rG compared with dG and 7,8-dihydro-8-oxo-2-deoxyguanosine (8-oxo-dG), whereas pol easily bypassed the ribonucleotides. pol ␣ exclusively inserted dAMP opposite 8-oxo-rG. Interestingly, pol preferentially inserted dCMP opposite 8-oxo-rG, whereas the insertion of dAMP was favored opposite 8-oxo-dG. In addition, pol accurately bypassed 8-oxo-rG. Furthermore, we examined the activity of the base excision repair (BER) enzymes 8-oxoguanine DNA glycosylase (OGG1) and apurinic/apyrimidinic endonuclease 1 on the substrates, including rG and 8-oxorG. Both BER enzymes were completely inactive against 8-oxo-rG in DNA. However, OGG1 suppressed 8-oxo-rG excision by RNase H2, which is involved in the removal of ribonucleotides from DNA. These results suggest that the different sugar backbones between 8-oxo-rG and 8-oxo-dG alter the capacity of TLS and repair of 8-oxoguanine.

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.

Background: Base excision repair is an important pathway for cytosine demethylation at the CpG dinucleotide for epigenetic control. Results: A deaminated 5-methylcytosine (thymine) and an adjacent oxidized guanine (7,8-dihydro-8-oxoguanine) retard base excision repair of each lesion. Conclusion: Altered in-tandem CpG dinucleotide is a poor substrate for base excision repair. Significance: Oxidized guanine in a CpG dinucleotide interferes with active DNA demethylation.

Human 8-oxoguanine DNA glycosylase (OGG1) excises the mutagenic oxidative DNA lesion 8-oxo-7,8-dihydroguanine (8-oxoG) from DNA. Kinetic characterization of OGG1 is undertaken to measure the rates of 8-oxoG excision and product release. When the OGG1 concentration is lower than substrate DNA, time courses of product formation are biphasic; a rapid exponential phase (i.e. burst) of product formation is followed by a linear steady-state phase. The initial burst of product formation corresponds to the concentration of enzyme properly engaged on the substrate, and the burst amplitude depends on the concentration of enzyme. The first-order rate constant of the burst corresponds to the intrinsic rate of 8-oxoG excision and the slower steady-state rate measures the rate of product release (product DNA dissociation rate constant, k(off)). Here, we describe steady-state, pre-steady-state, and single-turnover approaches to isolate and measure specific steps during OGG1 catalytic cycling. A fluorescent labeled lesion-containing oligonucleotide and purified OGG1 are used to facilitate precise kinetic measurements. Since low enzyme concentrations are used to make steady-state measurements, manual mixing of reagents and quenching of the reaction can be performed to ascertain the steady-state rate (k(off)). Additionally, extrapolation of the steady-state rate to a point on the ordinate at zero time indicates that a burst of product formation occurred during the first turnover (i.e. y-intercept is positive). The first-order rate constant of the exponential burst phase can be measured using a rapid mixing and quenching technique that examines the amount of product formed at short time intervals (<1 sec) before the steady-state phase and corresponds to the rate of 8-oxoG excision (i.e. chemistry). The chemical step can also be measured using a single-turnover approach where catalytic cycling is prevented by saturating substrate DNA with enzyme (E>S). These approaches can measure elementary rate constants that influence the efficiency of removal of a DNA lesion.

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 Polη and Polκ proteins that might modulate their specificity for incorporating 8-oxo-dGTP into DNA. We found that Arg61 in Polη 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 Polη) to 7 : 1 (R61K). Similarly, Tyr112 in Polκ was crucial for erroneous nucleotide incorporation. When Tyr112 was substituted with alanine (Y112A), the ratio of pairing was reduced from 11: 1 (wild-type Polκ) to almost 1: 1 (Y112A). Interestingly, substitution at the corresponding position in Polη, i.e. Phe18 to alanine, did not alter the specificity. These results suggested that amino acids at distinct positions in the active sites of Polη and Polκ might enhance 8-oxo-dGTP to favor the syn conformation, and thus direct its misincorporation into DNA.

Ribonucleotides, which are RNA precursors, are often incorporated into DNA during replication. Although embedded ribonucleotides in the genome are efficiently removed by canonical ribonucleotide excision repair (RER), inactivation of RER causes genomic ribonucleotide accumulation, leading to various abnormalities in cells. Mutation of genes encoding factors involved in RER is associated with the neuroinflammatory autoimmune disorder Aicardi–Goutières syndrome. Over the last decade, the biological impact of ribonucleotides in the genome has attracted much attention. In the present review, we particularly focus on recent studies that have elucidated possible mechanisms of ribonucleotide incorporation and repair and their significance in mammals.