Sloppy Bypass of an Abasic Lesion Catalyzed by a Y-family DNA Polymerase

Abstract

DNA damage that eludes cellular repair pathways can arrest the replication machinery and stall the cell cycle. However, this damage can be bypassed by the Y-family DNA polymerases. Here, Dpo4, an archetypal Y-family member from the thermophilic Sulfolobus solfataricus, was used to extend our kinetic studies of the bypass of an abasic site, one of the most mutagenic and ubiquitous cellular lesions. A short oligonucleotide sequencing assay is developed to directly sequence DNA bypass products synthesized by Dpo4. Our results show that incorporation upstream of the abasic lesion is replicated error-free; yet dramatically, once Dpo4 encounters the lesion, synthesis became sloppy, with bypass products containing a myriad of mutagenic events. Incorporation of dAMP (29%) and dCMP (53%) opposite the abasic lesion at 37°C correlates exceptionally well with our kinetic results and demonstrates two dominant bypass pathways via the A-rule and the lesion loop-out mechanism. Interestingly, the percentage of overall frameshift mutations increased from 71 (37°C) to 87% (75°C). Further analysis indicates that lesion bypass via the A-rule is strongly preferred over the lesion loop-out mechanism at higher temperatures and concomitantly reduces the occurrence of "؊1 deletion" mutations observed opposite the lesion at lower temperatures. The bypass percentage via the latter pathway is confirmed by an enzymatic digestion assay, verifying the reliability of our sequencing assay. Our results demonstrate that an abasic lesion causes Dpo4 and possibly all Y-family members to switch from a normal to a very mutagenic mode of replication.Cellular DNA is continually damaged by agents both endogenous and exogenous to an organism. This damage structurally modifies DNA, arresting replication by stalling the replicative DNA polymerase. The inability to copy the genome leads to cell cycle stalling and possibly cell death. Although a large portion of DNA lesions is restored by cellular repair pathways, a biologically significant amount of damage persists. Fortunately, organisms have evolved specialized enzymes known as the Y-family DNA polymerases that are capable of replicating damaged DNA in either an error-free or error-prone fashion (1, 2), thus rescuing a stalled replisome.Although the Y-family DNA polymerases collectively synthesize one to a few nucleotides per binding event, they all lack proofreading exonuclease activity and generate up to 100,000-fold more errors on undamaged DNA when compared with the astonishingly accurate replicative DNA polymerases (1, 3, 4). This high error rate is due to the loose active sites in the structures of the eukaryotic polymerases: human polymerase (5), yeast polymerase (6), human polymerase (pol ) (7), and yeast Rev1 (8), as well as the thermophilic archaeal Sulfolobus solfataricus DNA polymerase IV (Dpo4) 3 (9) and Sulfolobus acidocaldarius Dbh (10). These more permissive active sites impart unique functional attributes to the Y-family, including the ability to replicate through aberrant and bulky DNA le...

Impact of Conformational Heterogeneity of OxoG Lesions and Their Pairing Partners on Bypass Fidelity by Y Family Polymerases

“…For example, it has been previously noted that there was inefficient misinsertion of dGTP opposite template T by Dpo4, and that the bases of either the incoming nucleotide or the 3′ nucleotide of the primer strand were disordered in the electron density maps, indicating conformational heterogeneity (Vaisman et al, 2005). The array of possibilities in the accommodation of an abasic site within the Dpo4 active site includes base deletion and addition frameshift structures (Ling et al, 2004a); this correlates with a ∼200-fold slower insertion and extension opposite an abasic site compared with an unmodified template (Fiala et al, 2007) and various mutagenic events observed in the fully extended products (Fiala and Suo, 2007). Conformational heterogeneity of lesions and their pairing partners, involving either a 3′-primer terminus or an incoming dNTP (Yang and Woodgate, 2007), creates a challenge to achieving a reaction-ready alignment of the spacious active site, thereby suggestive of a dynamic fidelity-check screening mechanism in Y-family polymerases.…”

Mechanistic Studies of the Bypass of a Bulky Single-base Lesion Catalyzed by a Y-family DNA Polymerase

“…Running Start Assay-The running start assay was performed as previously described (17,18,26). Briefly, a preincubated solution of 5Ј-32 P-labeled DNA (100 nM) and Dpo4 (100 nM) in buffer R was rapidly mixed with a solution containing all four dNTPs (200 M each) at 37°C via a rapid chemical-quench flow apparatus (KinTek).…”

Recent Insight into the Kinetic Mechanisms and Conformational Dynamics of Y-Family DNA Polymerases

“…103 This novel methodology was used to confirm the relative frequencies of insertion of dATP versus insertion of dCTP via the lesion loop-out mechanism predicted from the kinetic efficiencies of each pathway and to provide information about more rare mutation events both opposite and downstream from the lesion. 103 The development of “next-generation sequencing” allows for a high-throughput alternative to the original SOSA method and has been used to assess the mutagenic profiles of several carboxymethylated DNA lesions in E. coli .…”

“…103 This novel methodology was used to confirm the relative frequencies of insertion of dATP versus insertion of dCTP via the lesion loop-out mechanism predicted from the kinetic efficiencies of each pathway and to provide information about more rare mutation events both opposite and downstream from the lesion. 103 The development of “next-generation sequencing” allows for a high-throughput alternative to the original SOSA method and has been used to assess the mutagenic profiles of several carboxymethylated DNA lesions in E. coli . 104 In a recent publication, novel software (Next-Generation Position Base Counter, available for download at ) was developed to allow for efficient analysis of millions of DNA sequences yielded from the next-generation sequencing-based high-throughput version of the SOSA technique (HT-SOSA).…”