Mechanism of genome instability mediated by human DNA polymerase mu misincorporation.
Ontology highlight
ABSTRACT:
SUBMITTER: Guo M
PROVIDER: S-EPMC8213813 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Although a defect in the DNA polymerase POLQ leads to ionizing radiation sensitivity in mammalian cells, the relevant enzymatic pathway has not been identified. Here we define the specific mechanism by which POLQ restricts harmful DNA instability. Our experiments show that Polq-null murine cells are selectively hypersensitive to DNA strand breaking agents, and that damage resistance requires the DNA polymerase activity of POLQ. Using a DNA break end joining assay in cells, we monitored repair of DNA ends with long 3' single-stranded overhangs. End joining events retaining much of the overhang were dependent on POLQ, and independent of Ku70. To analyze the repair function in more detail, we examined immunoglobulin class switch joining between DNA segments in antibody genes. POLQ participates in end joining of a DNA break during immunoglobulin class-switching, producing insertions of base pairs at the joins with homology to IgH switch-region sequences. Biochemical experiments with purified human POLQ protein revealed the mechanism generating the insertions during DNA end joining, relying on the unique ability of POLQ to extend DNA from minimally paired primers. DNA breaks at the IgH locus can sometimes join with breaks in Myc, creating a chromosome translocation. We found a marked increase in Myc/IgH translocations in Polq-defective mice, showing that POLQ suppresses genomic instability and genome rearrangements originating at DNA double-strand breaks. This work clearly defines a role and mechanism for mammalian POLQ in an alternative end joining pathway that suppresses the formation of chromosomal translocations. Our findings depart from the prevailing view that alternative end joining processes are generically translocation-prone.
| S-EPMC4183433 | biostudies-literature
Project description:Microhomology-mediated end-joining (MMEJ) is an error-prone alternative double-strand break-repair pathway that uses sequence microhomology to recombine broken DNA. Although MMEJ has been implicated in cancer development, the mechanism of this pathway is unknown. We demonstrate that purified human DNA polymerase ? (Pol?) performs MMEJ of DNA containing 3' single-strand DNA overhangs with ?2 bp of homology, including DNA modeled after telomeres, and show that MMEJ is dependent on Pol? in human cells. Our data support a mechanism whereby Pol? facilitates end-joining and microhomology annealing, then uses the opposing overhang as a template in trans to stabilize the DNA synapse. Pol? exhibits a preference for DNA containing a 5'-terminal phosphate, similarly to polymerases involved in nonhomologous end-joining. Finally, we identify a conserved loop domain that is essential for MMEJ and higher-order structures of Pol? that probably promote DNA synapse formation.
| S-EPMC4351179 | biostudies-literature
Project description:Messenger RNA (mRNA) translation can lead to higher rates of mRNA decay, suggesting a role for the ribosome in mRNA destruction. Furthermore, features of an mRNA, such as codon identities, that are directly probed by the ribosome also correlate with mRNA decay rates. Specifically, many amino acids are encoded by synonymous codons, and some synonymous codons are decoded by more abundant tRNAs leading to more optimal translation and increased mRNA stability. In addition to different translation rates, the presence of individual codons can lead to higher or lower rates of amino acid misincorporation which could potentially lead to protein misfolding if an individual amino acid makes many critical contacts in a structure. Here, we directly test whether amino acid misincorporation affects mRNA stability, taking advantage of an aminoglycoside antibiotic (G418) which promotes higher error rates in the ribosome. We observe that G418 decreases firefly luciferase mRNA stability in an in vitro system, and we similarly observe that G418 reduces mRNA stability in mouse embryonic stem cells (mESCs). G418-sensitive mRNAs are enriched for suboptimal hydrophobic amino acid codons as well as other codons that are known to result in higher rates of amino acid misincorporation. Since protein folding is highly sensitive to the identity of hydrophobic amino acids, these results strongly suggest that defects in protein folding are linked to mRNA decay.
2022-03-01 | GSE184874 | GEO
Project description:The DNA polymerase zeta (Polζ) plays a critical role in bypassing DNA damage. REV3L, the catalytic subunit of Polζ is also essential in mouse embryonic development and cell proliferation for reasons that remain incompletely understood. In this study, we reveal that REV3L protein interacts with heterochromatin components including repressive histone marks, and localizes in pericentromeric regions through direct interaction with HP1 dimer. We demonstrate that Polζ/REV3L ensures progression of replication forks through difficult-to-replicate pericentromeric heterochromatin, thereby preventing spontaneous chromosome break formation. We also find that Rev3l-deficient cells are compromised in the repair of heterochromatin-associated double-strand breaks, eliciting deletions in late replicating regions. Lack of REV3L leads to further consequences that may be ascribed to heterochromatin replication and repair-associated functions of Polζ, with a disruption of the temporal replication program at specific loci. This is correlated with changes in epigenetic landscape and transcriptional control of developmentally regulated genes. These results reveal a new function of Polζ in preventing chromosome instability during replication of heterochromatic regions.
| S-SCDT-EMBOJ-2020-104543 | biostudies-other
Project description:The DNA polymerase zeta (Polζ) plays a critical role in bypassing DNA damage. REV3L, the catalytic subunit of Polζ, is also essential in mouse embryonic development and cell proliferation for reasons that remain incompletely understood. In this study, we reveal that REV3L protein interacts with heterochromatin components including repressive histone marks and localizes in pericentromeric regions through direct interaction with HP1 dimer. We demonstrate that Polζ/REV3L ensures progression of replication forks through difficult-to-replicate pericentromeric heterochromatin, thereby preventing spontaneous chromosome break formation. We also find that Rev3l-deficient cells are compromised in the repair of heterochromatin-associated double-stranded breaks, eliciting deletions in late-replicating regions. Lack of REV3L leads to further consequences that may be ascribed to heterochromatin replication and repair-associated functions of Polζ, with a disruption of the temporal replication program at specific loci. This is correlated with changes in epigenetic landscape and transcriptional control of developmentally regulated genes. These results reveal a new function of Polζ in preventing chromosome instability during replication of heterochromatic regions.
| S-EPMC8561639 | biostudies-literature
Project description:Expansions of simple DNA repeats cause numerous hereditary diseases in humans. We analyzed the role of DNA polymerases in the instability of Friedreich's ataxia (GAA)(n) repeats in a yeast experimental system. The elementary step of expansion corresponded to ~160 bp in the wild-type strain, matching the size of Okazaki fragments in yeast. This step increased when DNA polymerase ? was mutated, suggesting a link between the scale of expansions and Okazaki fragment size. Expandable repeats strongly elevated the rate of mutations at substantial distances around them, a phenomenon we call repeat-induced mutagenesis (RIM). Notably, defects in the replicative DNA polymerases ? and ? strongly increased rates for both repeat expansions and RIM. The increases in repeat-mediated instability observed in DNA polymerase ? mutants depended on translesion DNA polymerases. We conclude that repeat expansions and RIM are two sides of the same replicative mechanism.
| S-EPMC3513503 | biostudies-literature
Project description:A novel DNA polymerase (Pol mu) has been recently identified in human cells. The amino-acid sequence of Pol mu is 42% identical to that of terminal deoxynucleotidyl transferase (TdT), a DNA-independent DNA polymerase that contributes to antigen-receptor diversity. In this paper we review the evidence supporting the role of Pol mu in somatic hypermutation of immunoglobulin genes, a T-dependent process that selectively occurs at germinal centres: (i) preferential expression in secondary lymphoid organs; (ii) expression associated to developing germinal centres; and (iii) very low base discrimination during DNA-dependent DNA polymerization by Pol mu, a mutator phenotype enormously accentuated by the presence of activating Mn2+ ions. Moreover, its similarity to TdT, together with extrapolation to the crystal structure of DNA polymerase beta complexed (Pol beta) with DNA, allows us to discuss the structural basis for the unprecedented error proneness of Pol mu, and to predict that Pol mu is structurally well suited to participate also in DNA end-filling steps occurring both during V(D)J recombination and repair of DNA double-strand breaks that are processed by non-homologous end-joining.
| S-EPMC1087697 | biostudies-other
Project description:Recent studies have described a DNA damage tolerance pathway choice that involves a competition between PrimPol-mediated repriming and fork reversal. Screening different translesion DNA synthesis (TLS) polymerases by the use of tools for their depletion, we identified a unique role of Pol ι in regulating such a pathway choice. Pol ι deficiency unleashes PrimPol-dependent repriming, which accelerates DNA replication in a pathway that is epistatic with ZRANB3 knockdown. In Pol ι-depleted cells, the excess participation of PrimPol in nascent DNA elongation reduces replication stress signals, but thereby also checkpoint activation in S phase, triggering chromosome instability in M phase. This TLS-independent function of Pol ι requires its PCNA-interacting but not its polymerase domain. Our findings unravel an unanticipated role of Pol ι in protecting the genome stability of cells from detrimental changes in DNA replication dynamics caused by PrimPol.
| S-EPMC10104471 | biostudies-literature
Project description:Ribonucleotides and 2'-deoxyribonucleotides are the basic units for RNA and DNA, respectively, and the only difference is the extra 2'-OH group on the ribonucleotide sugar. Cellular rNTP concentrations are much higher than those of dNTP. When copying DNA, DNA polymerases not only select the base of the incoming dNTP to form a Watson-Crick pair with the template base but also distinguish the sugar moiety. Some DNA polymerases use a steric gate residue to prevent rNTP incorporation by creating a clash with the 2'-OH group. Y-family human DNA polymerase ? (hpol ?) is of interest because of its spacious active site (especially in the major groove) and tolerance of DNA lesions. Here, we show that hpol ? maintains base selectivity when incorporating rNTPs opposite undamaged DNA and the DNA lesions 7,8-dihydro-8-oxo-2'-deoxyguanosine and cyclobutane pyrimidine dimer but with rates that are 10(3)-fold lower than for inserting the corresponding dNTPs. X-ray crystal structures show that the hpol ? scaffolds the incoming rNTP to pair with the template base (dG) or 7,8-dihydro-8-oxo-2'-deoxyguanosine with a significant propeller twist. As a result, the 2'-OH group avoids a clash with the steric gate, Phe-18, but the distance between primer end and P? of the incoming rNTP increases by 1 Å, elevating the energy barrier and slowing polymerization compared with dNTP. In addition, Tyr-92 was identified as a second line of defense to maintain the position of Phe-18. This is the first crystal structure of a DNA polymerase with an incoming rNTP opposite a DNA lesion.
| S-EPMC4759156 | biostudies-literature
Project description:The variant form of the human syndrome xeroderma pigmentosum (XPV) is caused by a deficiency in DNA polymerase eta (Poleta), a DNA polymerase that enables replication through ultraviolet-induced pyrimidine dimers. Here we report high-resolution crystal structures of human Poleta at four consecutive steps during DNA synthesis through cis-syn cyclobutane thymine dimers. Poleta acts like a 'molecular splint' to stabilize damaged DNA in a normal B-form conformation. An enlarged active site accommodates the thymine dimer with excellent stereochemistry for two-metal ion catalysis. Two residues conserved among Poleta orthologues form specific hydrogen bonds with the lesion and the incoming nucleotide to assist translesion synthesis. On the basis of the structures, eight Poleta missense mutations causing XPV can be rationalized as undermining the molecular splint or perturbing the active-site alignment. The structures also provide an insight into the role of Poleta in replicating through D loop and DNA fragile sites.
| S-EPMC2899710 | biostudies-literature