Nucleotide selection by the Y-family DNA polymerase Dpo4 involves template translocation and misalignment

Y-family DNA polymerases play a crucial role in translesion DNA synthesis. Here, we have characterized the binding kinetics and conformational dynamics of the Y-family polymerase Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) using single-molecule fluorescence. We find that in the absence of dNTPs, the binary complex shuttles between two different conformations within ∼1 s. These data are consistent with prior crystal structures in which the nucleotide binding site is either occupied by the terminal base pair (preinsertion conformation) or empty following Dpo4 translocation by 1 base pair (insertion conformation). Most interestingly, on dNTP binding, only the insertion conformation is observed and the correct dNTP stabilizes this complex compared with the binary complex, whereas incorrect dNTPs destabilize it. However, if the n+1 template base is complementary to the incoming dNTP, a structure consistent with a misaligned template conformation is observed, in which the template base at the n position loops out. This structure provides evidence for a Dpo4 mutagenesis pathway involving a transient misalignment mechanism.     

Three Residues of the Interdomain Linker Determine the Conformation and Single-base Deletion Fidelity of Y-family Translesion Polymerases

Dpo4 and Dbh are from two closely related Sulfolobus species and are well studied archaeal homologues of pol IV, an error prone Y-family polymerase from Escherichia coli. Despite sharing 54% amino acid identity, these polymerases display distinct mutagenic and translesion specificities. Structurally, Dpo4 and Dbh adopt different conformations because of the difference in relative orientation of their N-terminal catalytic and C-terminal DNA binding domains. Using chimeric constructs of these two polymerases, we have previously demonstrated that the interdomain linker is a major determinant of polymerase conformation, base-substitution fidelity, and abasic-site translesion synthesis. Here we find that the interdomain linker also affects the single-base deletion frequency and the mispair extension efficiency of these polymerases. Exchanging just three amino acids in the linkers of Dbh and Dpo4 is sufficient to change the fidelity by up to 30-fold, predominantly by altering the rate of correct (but not incorrect) nucleotide incorporation. Additionally, from a 2.4 Å resolution crystal structure, we have found that the three linker amino acids from Dpo4 are sufficient to allow Dbh to adopt the standard conformation of Dpo4. Thus, a small region of the interdomain linker, located more than 11 Å away from the catalytic residues, determines the fidelity of these Y-family polymerases, by controlling the alignment of substrates at the active site.     

Mechanism of double-base lesion bypass catalyzed by a Y-family DNA polymerase

As a widely used anticancer drug, cis-diamminedichloroplatinum(II) (cisplatin) reacts with adjacent purine bases in DNA to form predominantly cis-[Pt(NH(3))(2){d(GpG)-N7(1),-N7(2)}] intrastrand cross-links. Drug resistance, one of the major limitations of cisplatin therapy, is partially due to the inherent ability of human Y-family DNA polymerases to perform translesion synthesis in the presence of DNA-distorting damage such as cisplatin-DNA adducts. To better understand the mechanistic basis of translesion synthesis contributing to cisplatin resistance, this study investigated the bypass of a single, site-specifically placed cisplatin-d(GpG) adduct by a model Y-family DNA polymerase, Sulfolobus solfataricus DNA polymerase IV (Dpo4). Dpo4 was able to bypass this double-base lesion, although, the incorporation efficiency of dCTP opposite the first and second cross-linked guanine bases was decreased by 72- and 860-fold, respectively. Moreover, the fidelity at the lesion decreased up to two orders of magnitude. The cisplatin-d(GpG) adduct affected six downstream nucleotide incorporations, but interestingly the fidelity was essentially unaltered. Biphasic kinetic analysis supported a universal kinetic mechanism for the bypass of DNA lesions catalyzed by various translesion DNA polymerases. In conclusion, if human Y-family DNA polymerases adhere to this bypass mechanism, then translesion synthesis by these error-prone enzymes is likely accountable for cisplatin resistance observed in cancer patients.     

Roles of the four DNA polymerases of the crenarchaeon Sulfolobus solfataricus and accessory proteins in DNA replication

The hyperthermophilic crenarchaeon Sulfolobus solfataricus P2 encodes three B-family DNA polymerase genes, B1 (Dpo1), B2 (Dpo2), and B3 (Dpo3), and one Y-family DNA polymerase gene, Dpo4, which are related to eukaryotic counterparts. Both mRNAs and proteins of all four DNA polymerases were constitutively expressed in all growth phases. Dpo2 and Dpo3 possessed very low DNA polymerase and 3' to 5' exonuclease activities in vitro. Steady-state kinetic efficiencies (k(cat)/K(m)) for correct nucleotide insertion by Dpo2 and Dpo3 were several orders of magnitude less than Dpo1 and Dpo4. Both the accessory proteins proliferating cell nuclear antigen and the clamp loader replication factor C facilitated DNA synthesis with Dpo3, as with Dpo1 and Dpo4, but very weakly with Dpo2. DNA synthesis by Dpo2 and Dpo3 was remarkably decreased by single-stranded binding protein, in contrast to Dpo1 and Dpo4. DNA synthesis in the presence of proliferating cell nuclear antigen, replication factor C, and single-stranded binding protein was most processive with Dpo1, whereas DNA lesion bypass was most effective with Dpo4. Both Dpo2 and Dpo3, but not Dpo1, bypassed hypoxanthine and 8-oxoguanine. Dpo2 and Dpo3 bypassed uracil and cis-syn cyclobutane thymine dimer, respectively. High concentrations of Dpo2 or Dpo3 did not attenuate DNA synthesis by Dpo1 or Dpo4. We conclude that Dpo2 and Dpo3 are much less functional and more thermolabile than Dpo1 and Dpo4 in vitro but have bypass activities across hypoxanthine, 8-oxoguanine, and either uracil or cis-syn cyclobutane thymine dimer, suggesting their catalytically limited roles in translesion DNA synthesis past deaminated, oxidized base lesions and/or UV-induced damage.     

Translesion synthesis: Y-family polymerases and the polymerase switch

Replicative DNA polymerases are blocked at DNA lesions. Synthesis past DNA damage requires the replacement of the replicative polymerase by one of a group of specialised translesion synthesis (TLS) polymerases, most of which belong to the Y-family. Each of these has different substrate specificities for different types of damage. In eukaryotes mono-ubiquitination of PCNA plays a crucial role in the switch from replicative to TLS polymerases at stalled forks. All the Y-family polymerases have ubiquitin binding sites that increase their binding affinity for ubiquitinated PCNA at the sites of stalled forks.     

Proliferating cell nuclear antigen-dependent coordination of the biological functions of human DNA polymerase iota

Y-family DNA polymerases are believed to facilitate the replicative bypass of damaged DNA in a process commonly referred to as translesion synthesis. With the exception of DNA polymerase eta (poleta), which is defective in humans with the Xeroderma pigmentosum variant (XP-V) phenotype, little is known about the cellular function(s) of the remaining human Y-family DNA polymerases. We report here that an interaction between human DNA polymerase iota (poliota) and the proliferating cell nuclear antigen (PCNA) stimulates the processivity of poliota in a template-dependent manner in vitro. Mutations in one of the putative PCNA-binding motifs (PIP box) of poliota or the interdomain connector loop of PCNA diminish the binding between poliota and PCNA and concomitantly reduce PCNA-dependent stimulation of poliota activity. Furthermore, although retaining its capacity to interact with poleta in vivo, the poliota-PIP box mutant fails to accumulate in replication foci. Thus, PCNA, acting as both a scaffold and a modulator of the different activities involved in replication, appears to recruit and coordinate replicative and translesion DNA synthesis polymerases to ensure genome integrity.     

Snapshots of a Y-family DNA polymerase in replication: substrate-induced conformational transitions and implications for fidelity of Dpo4

Y-family DNA polymerases catalyze translesion DNA synthesis over damaged DNA. Each Y-family polymerase has a polymerase core consisting of a palm, finger and thumb domain in addition to a fourth domain known as a little finger domain. It is unclear how each domain moves during nucleotide incorporation and what type of conformational changes corresponds to the rate-limiting step previously reported in kinetic studies. Here, we present three crystal structures of the prototype Y-family polymerase: apo-Dpo4 at 1.9 Å resolution, Dpo4-DNA binary complex and Dpo4-DNA-dTMP ternary complex at 2.2 Å resolution. Dpo4 undergoes dramatic conformational changes from the apo to the binary structures with a 131° rotation of the little finger domain relative to the polymerase core upon DNA binding. This DNA-induced conformational change is verified in solution by our tryptophan fluorescence studies. In contrast, the polymerase core retains the same conformation in all three conformationally distinct states. Particularly, the finger domain which is responsible for checking base pairing between the template base and an incoming nucleotide retains a rigid conformation. The inflexibility of the polymerase core likely contributes to the low fidelity of Dpo4, in addition to its loose and solvent-accessible active site. Interestingly, while the binary and ternary complexes of Dpo4 retain an identical global conformation, the aromatic side chains of two conserved tyrosines at the nucleotide-binding site change orientations between the binary and ternary structures. Such local conformational changes may correspond to the rate-limiting step in the mechanism of nucleotide incorporation. Together, the global and local conformational transitions observed in our study provide a structural basis for the distinct kinetic steps of a catalytic cycle of DNA polymerization performed by a Y-family polymerase.     

The Y-family DNA polymerase Dpo4 uses a template slippage mechanism to create single-base deletions

The Y-family polymerases help cells tolerate DNA damage by performing translesion synthesis, yet they also can be highly error prone. One distinctive feature of the DinB class of Y-family polymerases is that they make single-base deletion errors at high frequencies in repetitive sequences, especially those that contain two or more identical pyrimidines with a 5' flanking guanosine. Intriguingly, different deletion mechanisms have been proposed, even for two archaeal DinB polymerases that share 54% sequence identity and originate from two strains of Sulfolobus. To reconcile these apparent differences, we have characterized Dpo4 from Sulfolobus solfataricus using the same biochemical and crystallographic approaches that we have used previously to characterize Dbh from Sulfolobus acidocaldarius. In contrast to previous suggestions that Dpo4 uses a deoxynucleoside triphosphate (dNTP)-stabilized misalignment mechanism when creating single-base deletions, we find that Dpo4 predominantly uses a template slippage deletion mechanism when replicating repetitive DNA sequences, as was previously shown for Dbh. Dpo4 stabilizes the skipped template base in an extrahelical conformation between the polymerase and the little-finger domains of the enzyme. This contrasts with Dbh, in which the extrahelical base is stabilized against the surface of the little-finger domain alone. Thus, despite sharing a common deletion mechanism, these closely related polymerases use different contacts with the substrate to accomplish the same result.     

Quantitative analysis of the mutagenic potential of 1-aminopyrene-DNA adduct bypass catalyzed by Y-family DNA polymerases

N-(Deoxyguanosin-8-yl)-1-aminopyrene (dG(AP)) is the predominant nitro polyaromatic hydrocarbon product generated from the air pollutant 1-nitropyrene reacting with DNA. Previous studies have shown that dG(AP) induces genetic mutations in bacterial and mammalian cells. One potential source of these mutations is the error-prone bypass of dG(AP) lesions catalyzed by the low-fidelity Y-family DNA polymerases. To provide a comparative analysis of the mutagenic potential of the translesion DNA synthesis (TLS) of dG(AP), we employed short oligonucleotide sequencing assays (SOSAs) with the model Y-family DNA polymerase from Sulfolobus solfataricus, DNA Polymerase IV (Dpo4), and the human Y-family DNA polymerases eta (hPolη), kappa (hPolκ), and iota (hPolι). Relative to undamaged DNA, all four enzymes generated far more mutations (base deletions, insertions, and substitutions) with a DNA template containing a site-specifically placed dG(AP). Opposite dG(AP) and at an immediate downstream template position, the most frequent mutations made by the three human enzymes were base deletions and the most frequent base substitutions were dAs for all enzymes. Based on the SOSA data, Dpo4 was the least error-prone Y-family DNA polymerase among the four enzymes during the TLS of dG(AP). Among the three human Y-family enzymes, hPolκ made the fewest mutations at all template positions except opposite the lesion site. hPolκ was significantly less error-prone than hPolι and hPolη during the extension of dG(AP) bypass products. Interestingly, the most frequent mutations created by hPolι at all template positions were base deletions. Although hRev1, the fourth human Y-family enzyme, could not extend dG(AP) bypass products in our standing start assays, it preferentially incorporated dCTP opposite the bulky lesion. Collectively, these mutagenic profiles suggest that hPolk and hRev1 are the most suitable human Y-family DNA polymerases to perform TLS of dG(AP) in humans.     

Low fidelity DNA synthesis by a y family DNA polymerase due to misalignment in the active site

Sulfolobus solfataricus DNA polymerase IV (Dpo4) is a member of the Y family of DNA polymerases whose crystal structure has recently been solved. As a model for other evolutionarily conserved Y family members that perform translesion DNA synthesis and have low fidelity, we describe here the base substitution and frameshift fidelity of DNA synthesis by Dpo4. Dpo4 generates all 12 base-base mismatches at high rates, 11 of which are similar to those of its human homolog, DNA polymerase kappa. This result is consistent with the Dpo4 structure, implying lower geometric selection for correct base pairs. Surprisingly, Dpo4 generates C.dCMP mismatches at an unusually high average rate and preferentially at cytosine flanked by 5'-template guanine. Dpo4 also has very low frameshift fidelity and frequently generates deletions of even noniterated nucleotides, especially cytosine flanked by a 5'-template guanine. Both unusual features of error specificity suggest that Dpo4 can incorporate dNTP precursors when two template nucleotides are present in the active site binding pocket. These results have implications for mutagenesis resulting from DNA synthesis by Y family polymerases.     

Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4): an archaeal DinB-like DNA polymerase with lesion-bypass properties akin to eukaryotic polη

Phylogenetic analysis of Y-family DNA polymerases suggests that it can be subdivided into several discrete branches consisting of UmuC/DinB/Rev1/Rad30/Rad30A and Rad30B. The most diverse is the DinB family that is found in all three kingdoms of life. Searches of the complete genome of the crenarchaeon Sulfolobus solfataricus P2 reveal that it possesses a DinB homolog that has been termed DNA polymerase IV (Dpo4). We have overproduced and purified native Dpo4 protein and report here its enzymatic characterization. Dpo4 is thermostable, but can also synthesize DNA at 37°C. Under these conditions, the enzyme exhibits misinsertion fidelities in the range of 8 × 10^–3 to 3 × 10^–4. Dpo4 is distributive but at high enzyme to template ratios can synthesize long stretches of DNA and can substitute for Taq polymerase in PCR. On damaged DNA templates, Dpo4 can facilitate translesion replication of an abasic site, a cis-syn thymine–thymine dimer, as well as acetyl aminofluorene adducted- and cisplatinated-guanine residues. Thus, although phylogenetically related to DinB polymerases, our studies suggest that the archaeal Dpo4 enzyme exhibits lesion-bypass properties that are, in fact, more akin to those of eukaryotic polη.     

Structural basis of ubiquitin recognition by translesion synthesis DNA polymerase ι

Cells have evolved mutagenic bypass mechanisms that prevent stalling of the replication machinery at DNA lesions. This process, translesion DNA synthesis (TLS), involves switching from high-fidelity DNA polymerases to specialized DNA polymerases that replicate through a variety of DNA lesions. In eukaryotes, polymerase switching during TLS is regulated by the DNA damage-triggered monoubiquitylation of PCNA. How the switch operates is unknown, but all TLS polymerases of the so-called Y-family possess PCNA and ubiquitin-binding domains that are important for their function. To gain insight into the structural mechanisms underlying the regulation of TLS by ubiquitylation, we have probed the interaction of ubiquitin with a conserved ubiquitin-binding motif (UBM2) of Y-family polymerase Polι. Using NMR spectroscopy, we have determined the structure of a complex of human Polι UBM2 and ubiquitin, revealing a novel ubiquitin recognition fold consisting of two α-helices separated by a central trans-proline residue conserved in all UBMs. We show that, different from the majority of ubiquitin complexes characterized to date, ubiquitin residue Ile44 only plays a modest role in the association of ubiquitin with Polι UBM2. Instead, binding of UBM2 is centered on the recognition of Leu8 in ubiquitin, which is essential for the interaction.     

Crystal structure of a Y-family DNA polymerase in action: a mechanism for error-prone and lesion-bypass replication

Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) is a DinB homolog that belongs to the recently described Y-family of DNA polymerases, which are best characterized by their low-fidelity synthesis on undamaged DNA templates and propensity to traverse normally replication-blocking lesions. Crystal structures of Dpo4 in ternary complexes with DNA and an incoming nucleotide, either correct or incorrect, have been solved at 1.7 A and 2.1 A resolution, respectively. Despite a conserved active site and a hand-like configuration similar to all known polymerases, Dpo4 makes limited and nonspecific contacts with the replicating base pair, thus relaxing base selection. Dpo4 is also captured in the crystal translocating two template bases to the active site at once, suggesting a possible mechanism for bypassing thymine dimers.     

Ubiquitin-family modifications in the replication of DNA damage

The cell uses specialised Y-family DNA polymerases or damage avoidance mechanisms to replicate past damaged sites in DNA. These processes are under complex regulatory systems, which employ different types of post-translational modification. All the Y-family polymerases have ubiquitin binding domains that bind to mono-ubiquitinated PCNA to effect the switching from replicative to Y-family polymerase. Ubiquitination and de-ubiquitination of PCNA are tightly regulated. There is also evidence for another as yet unidentified ubiquitinated protein being involved in recruitment of Y-family polymerases to chromatin. Poly-ubiquitination of PCNA stimulates damage avoidance, and, at least in yeast, PCNA is SUMOylated to prevent unwanted recombination events at the replication fork. The Y-family polymerases themselves can be ubiquitinated and, in the case of DNA polymerase η, this results in the polymerase being excluded from chromatin.     

Kinetic analysis of correct nucleotide insertion by a Y-family DNA polymerase reveals conformational changes both prior to and following phosphodiester bond formation as detected by tryptophan fluorescence

The Sulfolobus solfataricus Y-family DNA polymerase Dpo4 is a model for translesion replication and has been used in the analysis of individual steps involved in catalysis. The role of conformational changes has not been clear. Introduction of Trp residues into the Trp-devoid wild-type protein provided fluorescence probes of these events, particularly in the case of mutants T239W and N188W. With both mutants, a rapid increase in Trp fluorescence was observed only in the case of normal base pairing (G:C), was saturable with respect to dCTP concentration, and occurred in the absence of phosphodiester bond formation. A subsequent decrease in the Trp fluorescence occurred when phosphodiester bond formation was permitted, and these rates were independent of the dCTP concentration. This step is relatively slow and is attributed to a conformational relaxation step occurring after pyrophosphate release, which was measured and shown to be fast in a separate experiment. The measured rate of release of DNA from Dpo4 was rapid and is not rate-limiting. Overall, the measurements provide a kinetic scheme for Dpo4 different than generally accepted for replicative polymerases or proposed for Dpo4 and other Y-family polymerases: the initial enzyme.DNA.dNTP complex undergoes a rapid (18 s(-1)), reversible (21 s(-1)) conformational change, followed by relatively rapid phosphodiester bond formation (11 s(-1)) and then fast release of pyrophosphate, followed by a rate-limiting relaxation of the active conformation (2 s(-1)) and then rapid DNA release, yielding an overall steady-state kcat of <1 s(-1).     

DNA lesion alters global conformational dynamics of Y-family DNA polymerase during catalysis

A major product of oxidative damage to DNA, 8-oxo-7,8-dihydro-2'-deoxyguanine (8-oxoG), can lead to genomic mutations if it is bypassed unfaithfully by DNA polymerases in vivo. However, our pre-steady-state kinetic studies show that DNA polymerase IV (Dpo4), a prototype Y-family enzyme from Sulfolobus solfataricus, can bypass 8-oxoG both efficiently and faithfully. For the first time, our stopped-flow FRET studies revealed that a DNA polymerase altered its synchronized global conformational dynamics in response to a DNA lesion. Relative to nucleotide incorporation into undamaged DNA, three of the four domains of Dpo4 undertook different conformational transitions during 8-oxoG bypass and the subsequent extension step. Moreover, the rapid translocation of Dpo4 along DNA induced by nucleotide binding was significantly hindered by the interactions between the embedded 8-oxoG and Dpo4 during the extension step. These results unprecedentedly demonstrate that a Y-family DNA polymerase employs different global conformational dynamics when replicating undamaged and damaged DNA.     

Translesion Synthesis of Abasic Sites by Yeast DNA Polymerase ?

Studies of replicative DNA polymerases have led to the generalization that abasic sites are strong blocks to DNA replication. Here we show that yeast replicative DNA polymerase ϵ bypasses a model abasic site with comparable efficiency to Pol η and Dpo4, two translesion polymerases. DNA polymerase ϵ also exhibited high bypass efficiency with a natural abasic site on the template. Translesion synthesis primarily resulted in deletions. In cases where only a single nucleotide was inserted, dATP was the preferred nucleotide opposite the natural abasic site. In contrast to translesion polymerases, DNA polymerase ϵ with 3′–5′ proofreading exonuclease activity bypasses only the model abasic site during processive synthesis and cannot reinitiate DNA synthesis. This characteristic may allow other pathways to rescue leading strand synthesis when stalled at an abasic site.     

Structural mechanism of ribonucleotide discrimination by a Y-family DNA polymerase

The ability of DNA polymerases to differentiate between ribonucleotides and deoxribonucleotides is fundamental to the accurate replication and maintenance of an organism's genome. The active sites of Y-family DNA polymerases are highly solvent accessible, yet these enzymes still maintain a high selectivity towards deoxyribonucleotides. Here, we biochemically demonstrate that a single active-site mutation (Y12A) in Dpo4, a model Y-family DNA polymerase, causes both a dramatic loss of ribonucleotide discrimination and a decrease in nucleotide incorporation efficiency. We also determined two ternary crystal structures of the Dpo4 Y12A mutant incorporating either dATP or ATP nucleotides opposite a template dT base. Interestingly, both dATP and ATP were hydrolyzed to dADP and ADP, respectively. In addition, the dADP and ADP molecules adopt a similar conformation and position at the polymerase active site to a ddADP molecule in the ternary crystal structure of wild-type Dpo4. The Y12A mutant loses stacking interactions with the deoxyribose of dNTP, which destabilizes the binding of incoming nucleotides. The mutation also opens a space to accommodate the 2′-OH group of the ribose of NTP in the polymerase active site. The structural change leads to the reduction in deoxynucleotide incorporation efficiency and allows ribonucleotide incorporation.     

Global Conformational Dynamics of a Y-Family DNA Polymerase during Catalysis

Replicative DNA polymerases are stalled by damaged DNA while the newly discovered Y-family DNA polymerases are recruited to rescue these stalled replication forks, thereby enhancing cell survival. The Y-family DNA polymerases, characterized by low fidelity and processivity, are able to bypass different classes of DNA lesions. A variety of kinetic and structural studies have established a minimal reaction pathway common to all DNA polymerases, although the conformational intermediates are not well defined. Furthermore, the identification of the rate-limiting step of nucleotide incorporation catalyzed by any DNA polymerase has been a matter of long debate. By monitoring time-dependent fluorescence resonance energy transfer (FRET) signal changes at multiple sites in each domain and DNA during catalysis, we present here a real-time picture of the global conformational transitions of a model Y-family enzyme: DNA polymerase IV (Dpo4) from Sulfolobus solfataricus. Our results provide evidence for a hypothetical DNA translocation event followed by a rapid protein conformational change prior to catalysis and a subsequent slow, post-chemistry protein conformational change. Surprisingly, the DNA translocation step was induced by the binding of a correct nucleotide. Moreover, we have determined the directions, rates, and activation energy barriers of the protein conformational transitions, which indicated that the four domains of Dpo4 moved in a synchronized manner. These results showed conclusively that a pre-chemistry conformational change associated with domain movements was too fast to be the rate-limiting step. Rather, the rearrangement of active site residues limited the rate of correct nucleotide incorporation. Collectively, the conformational dynamics of Dpo4 offer insights into how the inter-domain movements are related to enzymatic function and their concerted interactions with other proteins at the replication fork.