The 5'-phosphate enhances the DNA-binding and exonuclease activities of human mitochondrial genome maintenance exonuclease 1 (MGME1).

In higher eukaryotes, mitochondria play multiple roles in energy production, signaling, and biosynthesis. Mitochondria possess multiple copies of mitochondrial DNA (mtDNA), which encodes 37 genes that are essential for mitochondrial and cellular function. When mtDNA is challenged by endogenous and exogenous factors, mtDNA undergoes repair, degradation, and compensatory synthesis. mtDNA degradation is an emerging pathway in mtDNA damage response and maintenance. A key factor involved is the human mitochondrial genome maintenance exonuclease 1 (MGME1). Despite previous biochemical and functional studies, controversies exist regarding the polarity of MGME1-mediated DNA cleavage. Also, how DNA sequence may affect the activities of MGME1 remains elusive. Such information is not only fundamental to the understanding of MGME1 but critical for deciphering the mechanism of mtDNA degradation. Herein, we use quantitative assays to examine the effects of substrate structure and sequence on the DNA-binding and enzymatic activities of MGME1. We demonstrate that MGME1 binds to and cleaves from the 5'-end of single-stranded DNA substrates, especially in the presence of 5'-phosphate, which plays an important role in DNA binding and optimal cleavage by MGME1. In addition, MGME1 tolerates certain modifications at the terminal end, such as a 5'-deoxyribosephosphate intermediate formed in base excision repair. We show that MGME1 processes different sequences with varying efficiencies, with dT and dC sequences being the most and least efficiently digested, respectively. Our results provide insights into the enzymatic properties of MGME1 and a rationale for the coordination of MGME1 with the 3'-5' exonuclease activity of DNA polymerase γ in mtDNA degradation.

DNA polymerases η and κ bypass N2-guanine-O6-alkylguanine DNA alkyltransferase cross-linked DNA-peptides.

DNA-protein cross-links are formed when proteins become covalently trapped with DNA in the presence of exogenous or endogenous alkylating agents. If left unrepaired, they inhibit transcription as well as DNA unwinding during replication and may result in genome instability or even cell death. The DNA repair protein O6-alkylguanine DNA-alkyltransferase (AGT) is known to form DNA cross-links in the presence of the carcinogen 1,2-dibromoethane, resulting in G:C to T:A transversions and other mutations in both bacterial and mammalian cells. We hypothesized that AGT-DNA cross-links would be processed by nuclear proteases to yield peptides small enough to be bypassed by translesion (TLS) polymerases. Here, a 15-mer and a 36-mer peptide from the active site of AGT were cross-linked to the N2 position of guanine via conjugate addition of a thiol containing a peptide dehydroalanine moiety. Bypass studies with DNA polymerases (pols) η and κ indicated that both can accurately bypass the cross-linked DNA peptides. The specificity constant (kcat/Km) for steady-state incorporation of the correct nucleotide dCTP increased by 6-fold with human (h) pol κ and 3-fold with hpol η, with hpol η preferentially inserting nucleotides in the order dC > dG > dA > dT. LC-MS/MS analysis of the extension product also revealed error-free bypass of the cross-linked 15-mer peptide by hpol η. We conclude that a bulky 15-mer AGT peptide cross-linked to the N2 position of guanine can retard polymerization, but that overall fidelity is not compromised because only correct bases are inserted and extended.

Deoxyribozyme-based method for absolute quantification of N6-methyladenosine fractions at specific sites of RNA.

N6-Methyladenosine (m6A) is the most prevalent modified base in eukaryotic mRNA and long noncoding RNA. Although candidate sites for the m6A modification are identified at the transcriptomic level, methods for site-specific quantification of absolute m6A modification levels are still limited. Herein, we present a facile method implementing a deoxyribozyme, VMC10, which preferentially cleaves the unmodified RNA. We leveraged reverse transcription and real-time quantitative PCR along with key control experiments to quantify the methylation fraction of specific m6A sites. We validated the accuracy of this method with synthetic RNA in which methylation fractions ranged from 0 to 100% and applied our method to several endogenous sites that were previously identified in sequencing-based studies. This method provides a time- and cost-effective approach for absolute quantification of the m6A fraction at specific loci, with the potential for multiplexed quantifications, expanding the current toolkit for studying RNA modifications.

A non-canonical role for the DNA glycosylase NEIL3 in suppressing APE1 endonuclease-mediated ssDNA damage.

The DNA glycosylase NEIL3 has been implicated in DNA repair pathways including the base excision repair and the interstrand cross-link repair pathways via its DNA glycosylase and/or AP lyase activity, which are considered canonical roles of NEIL3 in genome integrity. Compared with the other DNA glycosylases NEIL1 and NEIL2, Xenopus laevis NEIL3 C terminus has two highly conserved zinc finger motifs containing GRXF residues (designated as Zf-GRF). It has been demonstrated that the minor AP endonuclease APE2 contains only one Zf-GRF motif mediating interaction with single-strand DNA (ssDNA), whereas the major AP endonuclease APE1 does not. It appears that the two NEIL3 Zf-GRF motifs (designated as Zf-GRF repeat) are dispensable for its DNA glycosylase and AP lyase activity; however, the potential function of the NEIL3 Zf-GRF repeat in genome integrity remains unknown. Here, we demonstrate evidence that the NEIL3 Zf-GRF repeat was associated with a higher affinity for shorter ssDNA than one single Zf-GRF motif. Notably, our protein-protein interaction assays show that the NEIL3 Zf-GRF repeat but not one Zf-GRF motif interacted with APE1 but not APE2. We further reveal that APE1 endonuclease activity on ssDNA but not on dsDNA is compromised by a NEIL3 Zf-GRF repeat, whereas one Zf-GRF motif within NEIL3 is not sufficient to prevent such activity of APE1. In addition, COMET assays show that excess NEIL3 Zf-GRF repeat reduces DNA damage in oxidative stress in Xenopus egg extracts. Together, our results suggest a noncanonical role of NEIL3 in genome integrity via its distinct Zf-GRF repeat in suppressing APE1 endonuclease-mediated ssDNA breakage.

Impact of 1,N6-ethenoadenosine, a damaged ribonucleotide in DNA, on translesion synthesis and repair.

Incorporation of ribonucleotides into DNA can severely diminish genome integrity. However, how ribonucleotides instigate DNA damage is poorly understood. In DNA, they can promote replication stress and genomic instability and have been implicated in several diseases. We report here the impact of the ribonucleotide rATP and of its naturally occurring damaged analog 1,N6-ethenoadenosine (1,N6-ϵrA) on translesion synthesis (TLS), mediated by human DNA polymerase η (hpol η), and on RNase H2-mediated incision. Mass spectral analysis revealed that 1,N6-ϵrA in DNA generates extensive frameshifts during TLS, which can lead to genomic instability. Moreover, steady-state kinetic analysis of the TLS process indicated that deoxypurines (i.e. dATP and dGTP) are inserted predominantly opposite 1,N6-ϵrA. We also show that hpol η acts as a reverse transcriptase in the presence of damaged ribonucleotide 1,N6-ϵrA but has poor RNA primer extension activities. Steady-state kinetic analysis of reverse transcription and RNA primer extension showed that hpol η favors the addition of dATP and dGTP opposite 1,N6-ϵrA. We also found that RNase H2 recognizes 1,N6-ϵrA but has limited incision activity across from this lesion, which can lead to the persistence of this detrimental DNA adduct. We conclude that the damaged and unrepaired ribonucleotide 1,N6-ϵrA in DNA exhibits mutagenic potential and can also alter the reading frame in an mRNA transcript because 1,N6-ϵrA is incompletely incised by RNase H2.

The DNA repair enzyme MUTYH potentiates cytotoxicity of the alkylating agent MNNG by interacting with abasic sites.

Higher expression of the human DNA repair enzyme MUTYH has previously been shown to be strongly associated with reduced survival in a panel of 24 human lymphoblastoid cell lines exposed to the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). The molecular mechanism of MUTYH-enhanced MNNG cytotoxicity is unclear, because MUTYH has a well-established role in the repair of oxidative DNA lesions. Here, we show in mouse embryonic fibroblasts (MEFs) that this MNNG-dependent phenotype does not involve oxidative DNA damage and occurs independently of both O6-methyl guanine adduct cytotoxicity and MUTYH-dependent glycosylase activity. We found that blocking of abasic (AP) sites abolishes higher survival of Mutyh-deficient (Mutyh-/-) MEFs, but this blockade had no additive cytotoxicity in WT MEFs, suggesting the cytotoxicity is due to MUTYH interactions with MNNG-induced AP sites. We found that recombinant mouse MUTYH tightly binds AP sites opposite all four canonical undamaged bases and stimulated apurinic/apyrimidinic endonuclease 1 (APE1)-mediated DNA incision. Consistent with these observations, we found that stable expression of WT, but not catalytically-inactive MUTYH, enhances MNNG cytotoxicity in Mutyh-/- MEFs and that MUTYH expression enhances MNNG-induced genomic strand breaks. Taken together, these results suggest that MUTYH enhances the rapid accumulation of AP-site intermediates by interacting with APE1, implicating MUTYH as a factor that modulates the delicate process of base-excision repair independently of its glycosylase activity.

Catalytic DNA: Scope, Applications, and Biochemistry of Deoxyribozymes.

The discovery of natural RNA enzymes (ribozymes) prompted the pursuit of artificial DNA enzymes (deoxyribozymes) by in vitro selection methods. A key motivation is the conceptual and practical advantages of DNA relative to proteins and RNA. Early studies focused on RNA-cleaving deoxyribozymes, and more recent experiments have expanded the breadth of catalytic DNA to many other reactions. Including modified nucleotides has the potential to widen the scope of DNA enzymes even further. Practical applications of deoxyribozymes include their use as sensors for metal ions and small molecules. Structural studies of deoxyribozymes are only now beginning; mechanistic experiments will surely follow. Following the first report 21 years ago, the field of deoxyribozymes has promise for both fundamental and applied advances in chemistry, biology, and other disciplines.

The abundant DNA adduct N7-methyl deoxyguanosine contributes to miscoding during replication by human DNA polymerase η.

Aside from abasic sites and ribonucleotides, the DNA adduct N7-methyl deoxyguanosine (N7 -CH3 dG) is one of the most abundant lesions in mammalian DNA. Because N7 -CH3 dG is unstable, leading to deglycosylation and ring-opening, its miscoding potential is not well-understood. Here, we employed a 2'-fluoro isostere approach to synthesize an oligonucleotide containing an analog of this lesion (N7 -CH3 2'-F dG) and examined its miscoding potential with four Y-family translesion synthesis DNA polymerases (pols): human pol (hpol) η, hpol κ, and hpol ι and Dpo4 from the archaeal thermophile Sulfolobus solfataricus We found that hpol η and Dpo4 can bypass the N7 -CH3 2'-F dG adduct, albeit with some stalling, but hpol κ is strongly blocked at this lesion site, whereas hpol ι showed no distinction with the lesion and the control templates. hpol η yielded the highest level of misincorporation opposite the adduct by inserting dATP or dTTP. Moreover, hpol η did not extend well past an N7-CH3 2'-F dG:dT mispair. MS-based sequence analysis confirmed that hpol η catalyzes mainly error-free incorporation of dC, with misincorporation of dA and dG in 5-10% of products. We conclude that N7-CH3 2'-F dG and, by inference, N7-CH3 dG have miscoding and mutagenic potential. The level of misincorporation arising from this abundant adduct can be considered as potentially mutagenic as a highly miscoding but rare lesion.

Human DNA polymerase η has reverse transcriptase activity in cellular environments.

Classical DNA and RNA polymerase (pol) enzymes have defined roles with their respective substrates, but several pols have been found to have multiple functions. We reported previously that purified human DNA pol η (hpol η) can incorporate both deoxyribonucleoside triphosphates (dNTPs) and ribonucleoside triphosphates (rNTPs) and can use both DNA and RNA as substrates. X-ray crystal structures revealed that two pol η residues, Phe-18 and Tyr-92, behave as steric gates to influence sugar selectivity. However, the physiological relevance of these phenomena has not been established. Here, we show that purified hpol η adds rNTPs to DNA primers at physiological rNTP concentrations and in the presence of competing dNTPs. When two rATPs were inserted opposite a cyclobutane pyrimidine dimer, the substrate was less efficiently cleaved by human RNase H2. Human XP-V fibroblast extracts, devoid of hpol η, could not add rNTPs to a DNA primer, but the expression of transfected hpol η in the cells restored this ability. XP-V cell extracts did not add dNTPs to DNA primers hybridized to RNA, but could when hpol η was expressed in the cells. HEK293T cell extracts could add dNTPs to DNA primers hybridized to RNA, but lost this ability if hpol η was deleted. Interestingly, a similar phenomenon was not observed when other translesion synthesis (TLS) DNA polymerases-hpol ι, κ, or ζ-were individually deleted. These results suggest that hpol η is one of the major reverse transcriptases involved in physiological processes in human cells.

Dynamic interactions of the homologous pairing 2 (Hop2)-meiotic nuclear divisions 1 (Mnd1) protein complex with meiotic presynaptic filaments in budding yeast.

Homologous recombination (HR) is a universally conserved DNA repair pathway that can result in the exchange of genetic material. In eukaryotes, HR has evolved into an essential step in meiosis. During meiosis many eukaryotes utilize a two-recombinase pathway. This system consists of Rad51 and the meiosis-specific recombinase Dmc1. Both recombinases have distinct activities during meiotic HR, despite being highly similar in sequence and having closely related biochemical activities, raising the question of how these two proteins can perform separate functions. A likely explanation for their differential regulation involves the meiosis-specific recombination proteins Hop2 and Mnd1, which are part of a highly conserved eukaryotic protein complex that participates in HR, albeit through poorly understood mechanisms. To better understand how Hop2-Mnd1 functions during HR, here we used DNA curtains in conjunction with single-molecule imaging to measure and quantify the binding of the Hop2-Mnd1 complex from Saccharomyces cerevisiae to recombination intermediates comprising Rad51- and Dmc1-ssDNA in real time. We found that yeast Hop2-Mnd1 bound rapidly to Dmc1-ssDNA filaments with high affinity and remained bound for ∼1.3 min before dissociating. We also observed that this binding interaction was highly specific for Dmc1 and found no evidence for an association of Hop2-Mnd1 with Rad51-ssDNA or RPA-ssDNA. Our findings provide new quantitative insights into the binding dynamics of Hop2-Mnd1 with the meiotic presynaptic complex. On the basis of these findings, we propose a model in which recombinase specificities for meiotic accessory proteins enhance separation of the recombinases' functions during meiotic HR.

The highly specific, cell cycle-regulated methyltransferase from Caulobacter crescentus relies on a novel DNA recognition mechanism.

Two DNA methyltransferases, Dam and ß-class cell cycle-regulated DNA methyltransferase (CcrM), are key mediators of bacterial epigenetics. CcrM from the bacterium Caulobacter crescentus (CcrM C. crescentus, methylates adenine at 5'-GANTC-3') displays 105-107-fold sequence discrimination against noncognate sequences. However, the underlying recognition mechanism is unclear. Here, CcrM C. crescentus activity was either improved or mildly attenuated with substrates having one to three mismatched bp within or adjacent to the recognition site, but only if the strand undergoing methylation is left unchanged. By comparison, single-mismatched substrates resulted in up to 106-fold losses of activity with α (Dam) and γ-class (M.HhaI) DNA methyltransferases. We found that CcrM C. crescentus has a greatly expanded DNA-interaction surface, covering six nucleotides on the 5' side and eight nucleotides on the 3' side of its recognition site. Such a large interface may contribute to the enzyme's high sequence fidelity. CcrM C. crescentus displayed the same sequence discrimination with single-stranded substrates, and a surprisingly large (>107-fold) discrimination against ssRNA was largely due to the presence of two or more riboses within the cognate (DNA) site but not outside the site. Results from C-terminal truncations and point mutants supported our hypothesis that the recently identified C-terminal, 80-residue segment is essential for dsDNA recognition but is not required for single-stranded substrates. CcrM orthologs from Agrobacterium tumefaciens and Brucella abortus share some of these newly discovered features of the C. crescentus enzyme, suggesting that the recognition mechanism is conserved. In summary, CcrM C. crescentus uses a previously unknown DNA recognition mechanism.

The crystal structure of the Helicobacter pylori LlaJI.R1 N-terminal domain provides a model for site-specific DNA binding.

Restriction modification systems consist of an endonuclease that cleaves foreign DNA site-specifically and an associated methyltransferase that protects the corresponding target site in the host genome. Modification-dependent restriction systems, in contrast, specifically recognize and cleave methylated and/or glucosylated DNA. The LlaJI restriction system contains two 5-methylcytosine (5mC) methyltransferases (LlaJI.M1 and LlaJI.M2) and two restriction proteins (LlaJI.R1 and LlaJI.R2). LlaJI.R1 and LlaJI.R2 are homologs of McrB and McrC, respectively, which in Escherichia coli function together as a modification-dependent restriction complex specific for 5mC-containing DNA. Lactococcus lactis LlaJI.R1 binds DNA site-specifically, suggesting that the LlaJI system uses a different mode of substrate recognition. Here we present the structure of the N-terminal DNA-binding domain of Helicobacter pylori LlaJI.R1 at 1.97-Å resolution, which adopts a B3 domain fold. Structural comparison to B3 domains in plant transcription factors and other restriction enzymes identifies key recognition motifs responsible for site-specific DNA binding. Moreover, biochemistry and structural modeling provide a rationale for how H. pylori LlaJI.R1 may bind a target site that differs from the 5-bp sequence recognized by other LlaJI homologs and identify residues critical for this recognition activity. These findings underscore the inherent structural plasticity of B3 domains, allowing recognition of a variety of substrates using the same structural core.

BAH domains and a histone-like motif in DNA methyltransferase 1 (DNMT1) regulate de novo and maintenance methylation in vivo.

DNA methyltransferase 1 (DNMT1) is a multidomain protein believed to be involved only in the passive transmission of genomic methylation patterns via maintenance methylation. The mechanisms that regulate DNMT1 activity and targeting are complex and poorly understood. We used embryonic stem (ES) cells to investigate the function of the uncharacterized bromo-adjacent homology (BAH) domains and the glycine-lysine (GK) repeats that join the regulatory and catalytic domains of DNMT1. We removed the BAH domains by means of a CRISPR/Cas9-mediated deletion within the endogenous Dnmt1 locus. The internally deleted protein failed to associate with replication foci during S phase in vivo and lost the ability to mediate maintenance methylation. The data indicate that ablation of the BAH domains causes DNMT1 to be excluded from replication foci even in the presence of the replication focus-targeting sequence (RFTS). The GK repeats resemble the N-terminal tails of histones H2A and H4 and are normally acetylated. Substitution of lysines within the GK repeats with arginines to prevent acetylation did not alter the maintenance activity of DNMT1 but unexpectedly activated de novo methylation of paternal imprinting control regions (ICRs) in mouse ES cells; maternal ICRs remained unmethylated. We propose a model under which DNMT1 deposits paternal imprints in male germ cells in an acetylation-dependent manner. These data reveal that DNMT1 responds to multiple regulatory inputs that control its localization as well as its activity and is not purely a maintenance methyltransferase but can participate in the de novo methylation of a small but essential compartment of the genome.

Differential assembly of Rous sarcoma virus tetrameric and octameric intasomes is regulated by the C-terminal domain and tail region of integrase.

Retrovirus integrase (IN) catalyzes the concerted integration of linear viral DNA ends into chromosomes. The atomic structures of five different retrovirus IN-DNA complexes, termed intasomes, have revealed varying IN subunit compositions ranging from tetramers to octamers, dodecamers, and hexadecamers. Intasomes containing two IN-associated viral DNA ends capable of concerted integration are termed stable synaptic complexes (SSC), and those formed with a viral/target DNA substrate representing the product of strand-transfer reactions are strand-transfer complexes (STC). Here, we investigated the mechanisms associated with the assembly of the Rous sarcoma virus SSC and STC. C-terminal truncations of WT IN (286 residues) indicated a role of the last 18 residues ("tail" region) in assembly of the tetrameric and octameric SSC, physically stabilized by HIV-1 IN strand-transfer inhibitors. Fine mapping through C-terminal truncations and site-directed mutagenesis suggested that at least three residues (Asp-268-Thr-270) past the last ß-strand in the C-terminal domain (CTD) are necessary for assembly of the octameric SSC. In contrast, the assembly of the octameric STC was independent of the last 18 residues of IN. Single-site substitutions in the CTD affected the assembly of the SSC, but not necessarily of the STC, suggesting that STC assembly may depend less on specific interactions of the CTD with viral DNA. Additionally, we demonstrate that trans-communication between IN dimer-DNA complexes facilitates the association of native long-terminal repeat (LTR) ends with partially defective LTR ends to produce a hybrid octameric SSC. The differential assembly of the tetrameric and octameric SSC improves our understanding of intasomes.

Polymerase θ-helicase efficiently unwinds DNA and RNA-DNA hybrids.

POLQ is a unique multifunctional replication and repair gene that encodes for a N-terminal superfamily 2 helicase and a C-terminal A-family polymerase. Although the function of the polymerase domain has been investigated, little is understood regarding the helicase domain. Multiple studies have reported that polymerase θ-helicase (Polθ-helicase) is unable to unwind DNA. However, it exhibits ATPase activity that is stimulated by single-stranded DNA, which presents a biochemical conundrum. In contrast to previous reports, we demonstrate that Polθ-helicase (residues 1-894) efficiently unwinds DNA with 3'-5' polarity, including DNA with 3' or 5' overhangs, blunt-ended DNA, and replication forks. Polθ-helicase also efficiently unwinds RNA-DNA hybrids and exhibits a preference for unwinding the lagging strand at replication forks, similar to related HELQ helicase. Finally, we find that Polθ-helicase can facilitate strand displacement synthesis by Polθ-polymerase, suggesting a plausible function for the helicase domain. Taken together, these findings indicate nucleic acid unwinding as a relevant activity for Polθ in replication repair.

A DNA functionalized porphyrinic metal-organic framework as a peroxidase mimicking catalyst for amperometric determination of the activity of T4 polynucleotide kinase.

An electrochemical method is described for the sensitive detection of the activity of the enzyme T4 polynucleotide kinase (PNK) by using a DNA functionalized porphyrinic metal-organic framework (L/(Fe-P)n-MOF). In the presence of PNK, the hairpin oligonucleotide (HP1) becomes phosphorylated, and the trigger is released by lambda exonuclease (λ exo). The trigger DNA hybridizes with hairpin probe (immobilized on the gold electrode) to form a nicking endonuclease cleavage site. Thus, a single-strand capture probe is employed to hybridize with L/(Fe-P)n-MOF. The (Fe-P)n-MOF is a peroxidase mimicking material with high catalytic efficiency. By using this amplification strategy, an electrochemical signal is procured that allows for the determination of T4 PNK in the 1.0 mU·mL-1 to 1.0 U·mL-1 with a detection limit of 0.62 mU·mL-1. The method is selective and can be used to screen for enzyme inhibitors. Conceivably, the (Fe-P)n-MOF can also be used to detect other analytes via its peroxidase-mimicking activity. Graphical abstract Schematic presentation of T4 polynucleotide kinase (PNK) detection. Two hairpin DNAs (HP) and a porphyrinic metal-organic framework with peroxidase-mimicking activity are used. The detection limit is 0.62 mU mL-1 with enzyme assisted signal amplification. This method is selective and can be used to screen for enzyme inhibitors.

The Human Ligase IIIα-XRCC1 Protein Complex Performs DNA Nick Repair after Transient Unwrapping of Nucleosomal DNA.

Reactive oxygen species generate potentially cytotoxic and mutagenic lesions in DNA, both between and within the nucleosomes that package DNA in chromatin. The vast majority of these lesions are subject to base excision repair (BER). Enzymes that catalyze the first three steps in BER can act at many sites in nucleosomes without the aid of chromatin-remodeling agents and without irreversibly disrupting the host nucleosome. Here we show that the same is true for a protein complex comprising DNA ligase IIIα and the scaffolding protein X-ray repair cross-complementing protein 1 (XRCC1), which completes the fourth and final step in (short-patch) BER. Using in vitro assembled nucleosomes containing discretely positioned DNA nicks, our evidence indicates that the ligase IIIα-XRCC1 complex binds to DNA nicks in nucleosomes only when they are exposed by periodic, spontaneous partial unwrapping of DNA from the histone octamer; that the scaffolding protein XRCC1 enhances the ligation; that the ligation occurs within a complex that ligase IIIα-XRCC1 forms with the host nucleosome; and that the ligase IIIα-XRCC1-nucleosome complex decays when ligation is complete, allowing the host nucleosome to return to its native configuration. Taken together, our results illustrate ways in which dynamic properties intrinsic to nucleosomes may contribute to the discovery and efficient repair of base damage in chromatin.

Metnase Mediates Loading of Exonuclease 1 onto Single Strand Overhang DNA for End Resection at Stalled Replication Forks.

Stalling at DNA replication forks generates stretches of single-stranded (ss) DNA on both strands that are exposed to nucleolytic degradation, potentially compromising genome stability. One enzyme crucial for DNA replication fork repair and restart of stalled forks in human is Metnase (also known as SETMAR), a chimeric fusion protein consisting of a su(var)3-9, enhancer-of-zeste and trithorax (SET) histone methylase and transposase nuclease domain. We previously showed that Metnase possesses a unique fork cleavage activity necessary for its function in replication restart and that its SET domain is essential for recovery from hydroxyurea-induced DNA damage. However, its exact role in replication restart is unclear. In this study, we show that Metnase associates with exonuclease 1 (Exo1), a 5'-exonuclease crucial for 5'-end resection to mediate DNA processing at stalled forks. Metnase DNA cleavage activity was not required for Exo1 5'-exonuclease activity on the lagging strand daughter DNA, but its DNA binding activity mediated loading of Exo1 onto ssDNA overhangs. Metnase-induced enhancement of Exo1-mediated DNA strand resection required the presence of these overhangs but did not require Metnase's DNA cleavage activity. These results suggest that Metnase enhances Exo1-mediated exonuclease activity on the lagging strand DNA by facilitating Exo1 loading onto a single strand gap at the stalled replication fork.

Replisome-mediated translesion synthesis by a cellular replicase.

Genome integrity relies on the ability of the replisome to navigate ubiquitous DNA damage during DNA replication. The Escherichia coli replisome transiently stalls at leading-strand template lesions and can either reinitiate replication downstream of the lesion or recruit specialized DNA polymerases that can bypass the lesion via translesion synthesis. Previous results had suggested that the E. coli replicase might play a role in lesion bypass, but this possibility has not been tested in reconstituted DNA replication systems. We report here that the DNA polymerase III holoenzyme in a stalled E. coli replisome can directly bypass a single cyclobutane pyrimidine dimer or abasic site by translesion synthesis in the absence of specialized translesion synthesis polymerases. Bypass efficiency was proportional to deoxynucleotide concentrations equivalent to those found in vivo and was dependent on the frequency of primer synthesis downstream of the lesion. Translesion synthesis came at the expense of lesion-skipping replication restart. Replication of a cyclobutane pyrimidine dimer was accurate, whereas replication of an abasic site resulted in mainly -1 frameshifts. Lesion bypass was accompanied by an increase in base substitution frequency for the base preceding the lesion. These findings suggest that DNA damage at the replication fork can be replicated directly by the replisome without the need to activate error-prone pathways.

Human DNA polymerase η accommodates RNA for strand extension.

Ribonucleotides are the natural analogs of deoxyribonucleotides, which can be misinserted by DNA polymerases, leading to the most abundant DNA lesions in genomes. During replication, DNA polymerases tolerate patches of ribonucleotides on the parental strands to different extents. The majority of human DNA polymerases have been reported to misinsert ribonucleotides into genomes. However, only PrimPol, DNA polymerase α, telomerase, and the mitochondrial human DNA polymerase (hpol) γ have been shown to tolerate an entire RNA strand. Y-family hpol η is known for translesion synthesis opposite the UV-induced DNA lesion cyclobutane pyrimidine dimer and was recently found to incorporate ribonucleotides into DNA. Here, we report that hpol η is able to bind DNA/DNA, RNA/DNA, and DNA/RNA duplexes with similar affinities. In addition, hpol η, as well as another Y-family DNA polymerase, hpol κ, accommodates RNA as one of the two strands during primer extension, mainly by inserting dNMPs opposite unmodified templates or DNA lesions, such as 8-oxo-2'-deoxyguanosine or cyclobutane pyrimidine dimer, even in the presence of an equal amount of the DNA/DNA substrate. The discovery of this RNA-accommodating ability of hpol η redefines the traditional concept of human DNA polymerases and indicates potential new functions of hpol η in vivo.