Structure of human CST-pol-α/primase bound to a telomeric overhang poised for initiation of telomere C-strand synthesis

Telomere replication and regulation protect mammalian chromosome ends and promote genome stability. An essential step in telomere maintenance is the C-strand fill-in process, which is the de novo synthesis of the complementary strand of the telomere overhang. This step is catalyzed by polymerase-alpha/primase complex (pol-α/primase) and coordinated by an accessory factor, CTC1-STN1-TEN1 (CST). Using cryogenic-electron microscopy single-particle analysis, we report the structure of the human telomere C-strand fill-in preinitiation complex (PIC) at 3.9 Å resolution. The structure reveals a CST and a pol-α/primase co-bound to a single telomere overhang, poised for de novo RNA primer synthesis. Upon PIC assembly, the pol-α/primase undergoes large conformation change from its apo-state; CST partitions the DNA and RNA catalytic centers of pol-α/primase into two separate domains and positions the 3' end of an extended telomere single-stranded DNA template towards the RNA catalytic center (PRIM1 or p49). The telomeric single-stranded DNA template is further positioned by the POLA1 (or p180) catalytically dead exonuclease domain. Together with CST, the exonuclease domain forms a tight-fit molecular tunnel for template direction. Given the structural homology of CST to Replication Protein A (RPA), our structure provides the structural basis for a new model of how pol-α/primase lagging-strand DNA synthesis is coordinated by single-stranded DNA-binding accessory factors.

Additive effects of variants of unknown significance in replication repair-associated DNA polymerase genes on mutational burden and prognosis across diverse cancers

BackgroundDefects in replication repair-associated DNA polymerases often manifest an ultra-high tumor mutational burden (TMB), which is associated with higher probabilities of response to immunotherapies. The functional and clinical implications of different polymerase variants remain unclear.MethodsTargeted next-generation sequencing using a 425-cancer gene panel, which covers all exonic regions of three polymerase genes (POLE, POLD1, and POLH), was conducted in a cohort of 12,266 patients across 16 different tumor types from January 2017 to January 2019. Prognostication of POL variant-positive patients was performed using a cohort of 4679 patients from the The Cancer Genome Atlas (TCGA) datasets.ResultsThe overall prevalence of somatic and germline polymerase variants was 4.2% (95% CI 3.8% to 4.5%) and 0.7% (95% CI 0.5% to 0.8%), respectively, with highest frequencies in endometrial, urinary, prostate, and colorectal cancers (CRCs). While most germline polymerase variants showed no clear functional consequences, we identified a candidate p.T466A affecting the exonuclease domain of POLE, which might be underlying the early onset in a case with childhood CRC. Low frequencies of known hot-spot somatic mutations in POLE were detected and were associated with younger age, the male sex, and microsatellite stability. In both the panel and TCGA cohorts, POLE drivers exhibited high frequencies of alterations in genes in the DNA damage and repair (DDR) pathways, including BRCA2, ATM, MSH6, and ATR. Variants of unknown significance (VUS) of different polymerase domains showed variable penetrance with those in the exonuclease domain of POLE and POLD1 displaying high TMB. VUS in POL genes exhibited an additive effect as carriers of multiple VUS had exponentially increased TMB and prolonged overall survival. Similar to cases with driver mutations, the TMB-high POL VUS samples showed DDR pathway involvement and polymerase hypermutation signatures. Combinatorial analysis of POL and DDR pathway status further supported the potential additive effects of POL VUS and DDR pathway genes and revealed distinct prognostic subclasses that were independent of cancer type and TMB.ConclusionsOur results demonstrate the pathogenicity and additive prognostic value of POL VUS and DDR pathway gene alterations and suggest that genetic testing may be warranted in patients with diverse solid tumors.

Structure of an open conformation of T7 DNA Polymerase reveals novel structural features regulating primer-template stabilization at the polymerization active-site

The crystal structure of full-length T7 DNA polymerase in complex with its processivity factor thioredoxin and double-stranded DNA in the polymerization active site exhibits two novel structural motifs in family-A DNA polymerases: an extended b-hairpin at the fingers subdomain, that interacts with the DNA template strand downstream the primer-terminus, and a helix-loop-helix motif (insertion1) located between residues 102 to 122 in the exonuclease domain. The extended b-hairpin is involved in nucleotide incorporation on substrates with 5'-overhangs longer than 2 nucleotides, suggesting a role in stabilizing the template strand into the polymerization domain. Our biochemical data reveal that insertion1 of the exonuclease domain makes stabilizing interactions that facilitate proofreading by shuttling the primer strand into the exonuclease active site. Overall, our studies evidence conservation of the 3'-5' exonuclease domain fold between family-A DNA polymerases and highlight the modular architecture of T7 DNA polymerase. Our data suggest that the intercalating b-hairpin guides the template-strand into the polymerization active site after the T7 primase-helicase unwinds the DNA double helix ameliorating the formation of secondary structures and decreasing the appearance of indels

Possible changes in fidelity of DNA polymerase δ in ancestral mammals

DNA polymerase δ (polδ) is one of the major DNA polymerases that replicate chromosomal genomes in eukaryotes. Given the essential role of this protein, its phylogenetic tree was expected to reflect the relationship between taxa, like many other essential proteins. However, the tree of the catalytic subunit of polδ showed an unexpectedly strong heterogeneity among vertebrate lineages in evolutionary rate at the amino acid level, suggesting unusual amino acid substitutions specifically in the ancestral mammalian lineage. Structural and phylogenetic analyses were used to pinpoint where and when these amino acid substitutions occurred: around the 3′-5′ exonuclease domain in later mammal ancestry, after the split between monotremes and therians. The 3′-5′ exonuclease domain of this protein is known to have an impact on the fidelity of replication. Based on these observations, we explored the possibility that the amino acid substitutions we identified in polδ affected the mutation rate of entire chromosomal genomes in this time period.

Mutagenic mechanisms of cancer-associated DNA polymerase ε alleles

ABSTRACTA single amino acid residue change in the exonuclease domain of human DNA polymerase ε, P286R, is associated with the development of colorectal cancers, and has been shown to impart a mutagenic phenotype. Perhaps unexpectedly, the corresponding Pol ε allele in the yeast Saccharomyces cerevisiae (pol2-P301R), was found to drive greater mutagenesis than exonuclease-deficient Pol ε (pol2-4), a phenotype sometimes termed ultra-mutagenesis. By studying the impact on mutation frequency, type, replication-strand bias, and sequence context, we show that ultra-mutagenesis is commonly observed in cells carrying a range of cancer-associated Pol ε exonuclease domain alleles. Similarities between mutations generated by these alleles and those generated in pol2-4 cells indicate a shared mechanism of mutagenesis that yields a mutation pattern similar to cancer Signature 14. Comparison of POL2 ultra-mutator with pol2-M644G, a mutant in the polymerase domain decreasing Pol ε fidelity, revealed unexpected analogies in the sequence context and strand bias of mutations. Analysis of mutational patterns unique to exonuclease domain mutant cells suggests that backtracking of the polymerase, when the mismatched primer end cannot be accommodated in the proofreading domain, results in the observed increase in insertions and T>A mutations in specific sequence contexts.

Understanding the Effect of Multiple Domain Deletion in DNA Polymerase I from Geobacillus Sp. Strain SK72

The molecular structure of DNA polymerase I or family A polymerases is made up of three major domains that consist of a single polymerase domain with two extra exonuclease domains. When the N-terminal was deleted, the enzyme was still able to perform basic polymerase activity with additional traits that used isothermal amplification. However, the 3′-5′ exonuclease domain that carries a proofreading activity was disabled. Yet, the structure remained attached to the 5′-3′ polymerization domain without affecting its ability. The purpose of this non-functional domain still remains scarce. It either gives negative effects or provides structural support to the DNA polymerase. Here, we compared the effect of deleting each domain against the polymerase activity. The recombinant wild type and its variants were successfully purified and characterized. Interestingly, SK72-Exo (a large fragment excluding the 5′-3′ exonuclease domain) exhibited better catalytic activity than the native SK72 (with all three domains) at similar optimum temperature and pH profile, and it showed longer stability at 70 °C. Meanwhile, SK72-Exo2 (polymerization domain without both the 5′-3′ and 3′-5′ exonuclease domain) displayed the lowest activity with an optimum at 40 °C and favored a more neutral environment. It was also the least stable among the variants, with almost no activity at 50 °C for the first 10 min. In conclusion, cutting both exonuclease domains in DNA polymerase I has a detrimental effect on the polymerization activity and structural stability.