Association of DNA Polymerase μ (pol μ) with Ku and Ligase IV: Role for pol μ in End-Joining Double-Strand Break Repair

ABSTRACT Mammalian DNA polymerase μ (pol μ) is related to terminal deoxynucleotidyl transferase, but its biological role is not yet clear. We show here that after exposure of cells to ionizing radiation (IR), levels of pol μ protein increase. pol μ also forms discrete nuclear foci after IR, and these foci are largely coincident with IR-induced foci of γH2AX, a previously characterized marker of sites of DNA double-strand breaks. pol μ is thus part of the cellular response to DNA double-strand breaks. pol μ also associates in cell extracts with the nonhomologous end-joining repair factor Ku and requires both Ku and another end-joining factor, XRCC4-ligase IV, to form a stable complex on DNA in vitro. pol μ in turn facilitates both stable recruitment of XRCC4-ligase IV to Ku-bound DNA and ligase IV-dependent end joining. In contrast, the related mammalian DNA polymerase β does not form a complex with Ku and XRCC4-ligase IV and is less effective than pol μ in facilitating joining mediated by these factors. Our data thus support an important role for pol μ in the end-joining pathway for repair of double-strand breaks.

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Ku Heterodimer-Independent End Joining in Trypanosoma brucei Cell Extracts Relies upon Sequence Microhomology

Eukaryotic Cell ◽

10.1128/ec.00212-07 ◽

2007 ◽

Vol 6 (10) ◽

pp. 1773-1781 ◽

Cited By ~ 38

Author(s):

Peter Burton ◽

David J. McBride ◽

Jonathan M. Wilkes ◽

J. David Barry ◽

Richard McCulloch

Keyword(s):

Homologous Recombination ◽

Trypanosoma Brucei ◽

Antigenic Variation ◽

Bacterial Species ◽

Nonhomologous End Joining ◽

Dna Double Strand Breaks ◽

End Joining ◽

Strand Breaks ◽

Cell Extracts ◽

Ligase Iv

ABSTRACT DNA double-strand breaks (DSBs) are repaired primarily by two distinct pathways: homologous recombination and nonhomologous end joining (NHEJ). NHEJ has been found in all eukaryotes examined to date and has been described recently for some bacterial species, illustrating its ancestry. Trypanosoma brucei is a divergent eukaryotic protist that evades host immunity by antigenic variation, a process in which homologous recombination plays a crucial function. While homologous recombination has been examined in some detail in T. brucei, little work has been done to examine what other DSB repair pathways the parasite utilizes. Here we show that T. brucei cell extracts support the end joining of linear DNA molecules. These reactions are independent of the Ku heterodimer, indicating that they are distinct from NHEJ, and are guided by sequence microhomology. We also demonstrate bioinformatically that T. brucei, in common with other kinetoplastids, does not encode recognizable homologues of DNA ligase IV or XRCC4, suggesting that NHEJ is either absent or mechanistically diverged in these pathogens.

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Unexpected behavior of DNA polymerase Mu opposite template 8-oxo-7,8-dihydro-2′-guanosine

Nucleic Acids Research ◽

10.1093/nar/gkz680 ◽

2019 ◽

Vol 47 (17) ◽

pp. 9410-9422 ◽

Cited By ~ 1

Author(s):

Andrea M Kaminski ◽

Kishore K Chiruvella ◽

Dale A Ramsden ◽

Thomas A Kunkel ◽

Katarzyna Bebenek ◽

...

Keyword(s):

Double Strand Breaks ◽

Nonhomologous End Joining ◽

Dna Double Strand Breaks ◽

End Joining ◽

Strand Breaks ◽

Steady State Kinetics ◽

Ligase Iv ◽

Template Dna ◽

Dna Substrate

Abstract DNA double-strand breaks (DSBs) resulting from reactive oxygen species generated by exposure to UV and ionizing radiation are characterized by clusters of lesions near break sites. Such complex DSBs are repaired slowly, and their persistence can have severe consequences for human health. We have therefore probed DNA break repair containing a template 8-oxo-7,8-dihydro-2′-guanosine (8OG) by Family X Polymerase μ (Pol μ) in steady-state kinetics and cell-based assays. Pol μ tolerates 8OG-containing template DNA substrates, and the filled products can be subsequently ligated by DNA Ligase IV during Nonhomologous end-joining. Furthermore, Pol μ exhibits a strong preference for mutagenic bypass of 8OG by insertion of adenine. Crystal structures reveal that the template 8OG is accommodated in the Pol μ active site with none of the DNA substrate distortions observed for Family X siblings Pols β or λ. Kinetic characterization of template 8OG bypass indicates that Pol μ inserts adenosine nucleotides with weak sugar selectivity and, given the high cellular concentration of ATP, likely performs its role in repair of complex 8OG-containing DSBs using ribonucleotides.

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DNA polymerases δ and λ cooperate in repairing double-strand breaks by microhomology-mediated end-joining in Saccharomyces cerevisiae

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1507833112 ◽

2015 ◽

Vol 112 (50) ◽

pp. E6907-E6916 ◽

Cited By ~ 16

Author(s):

Damon Meyer ◽

Becky Xu Hua Fu ◽

Wolf-Dietrich Heyer

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Polymerase ◽

Genome Stability ◽

Double Strand Breaks ◽

Dna Polymerase Delta ◽

Dna Double Strand Breaks ◽

End Joining ◽

Strand Breaks ◽

Repair Efficiency ◽

Repair Pathways

Maintenance of genome stability is carried out by a suite of DNA repair pathways that ensure the repair of damaged DNA and faithful replication of the genome. Of particular importance are the repair pathways, which respond to DNA double-strand breaks (DSBs), and how the efficiency of repair is influenced by sequence homology. In this study, we developed a genetic assay in diploid Saccharomyces cerevisiae cells to analyze DSBs requiring microhomologies for repair, known as microhomology-mediated end-joining (MMEJ). MMEJ repair efficiency increased concomitant with microhomology length and decreased upon introduction of mismatches. The central proteins in homologous recombination (HR), Rad52 and Rad51, suppressed MMEJ in this system, suggesting a competition between HR and MMEJ for the repair of a DSB. Importantly, we found that DNA polymerase delta (Pol δ) is critical for MMEJ, independent of microhomology length and base-pairing continuity. MMEJ recombinants showed evidence that Pol δ proofreading function is active during MMEJ-mediated DSB repair. Furthermore, mutations in Pol δ and DNA polymerase 4 (Pol λ), the DNA polymerase previously implicated in MMEJ, cause a synergistic decrease in MMEJ repair. Pol λ showed faster kinetics associating with MMEJ substrates following DSB induction than Pol δ. The association of Pol δ depended on RAD1, which encodes the flap endonuclease needed to cleave MMEJ intermediates before DNA synthesis. Moreover, Pol δ recruitment was diminished in cells lacking Pol λ. These data suggest cooperative involvement of both polymerases in MMEJ.

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Altered DNA ligase activity in human disease

Mutagenesis ◽

10.1093/mutage/gez026 ◽

2019 ◽

Vol 35 (1) ◽

pp. 51-60 ◽

Cited By ~ 3

Author(s):

Alan E Tomkinson ◽

Tasmin Naila ◽

Seema Khattri Bhandari

Keyword(s):

Dna Damage ◽

Dna Replication ◽

Double Strand Breaks ◽

Dna Ligase ◽

Dna Double Strand Breaks ◽

End Joining ◽

Strand Breaks ◽

Dna Ligases ◽

Ligase Iv ◽

Non Homologous End Joining

Abstract The joining of interruptions in the phosphodiester backbone of DNA is critical to maintain genome stability. These breaks, which are generated as part of normal DNA transactions, such as DNA replication, V(D)J recombination and meiotic recombination as well as directly by DNA damage or due to DNA damage removal, are ultimately sealed by one of three human DNA ligases. DNA ligases I, III and IV each function in the nucleus whereas DNA ligase III is the sole enzyme in mitochondria. While the identification of specific protein partners and the phenotypes caused either by genetic or chemical inactivation have provided insights into the cellular functions of the DNA ligases and evidence for significant functional overlap in nuclear DNA replication and repair, different results have been obtained with mouse and human cells, indicating species-specific differences in the relative contributions of the DNA ligases. Inherited mutations in the human LIG1 and LIG4 genes that result in the generation of polypeptides with partial activity have been identified as the causative factors in rare DNA ligase deficiency syndromes that share a common clinical symptom, immunodeficiency. In the case of DNA ligase IV, the immunodeficiency is due to a defect in V(D)J recombination whereas the cause of the immunodeficiency due to DNA ligase I deficiency is not known. Overexpression of each of the DNA ligases has been observed in cancers. For DNA ligase I, this reflects increased proliferation. Elevated levels of DNA ligase III indicate an increased dependence on an alternative non-homologous end-joining pathway for the repair of DNA double-strand breaks whereas elevated level of DNA ligase IV confer radioresistance due to increased repair of DNA double-strand breaks by the major non-homologous end-joining pathway. Efforts to determine the potential of DNA ligase inhibitors as cancer therapeutics are on-going in preclinical cancer models.

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Persistent DNA damage signaling and DNA polymerase theta promote broken chromosome segregation

The Journal of Cell Biology ◽

10.1083/jcb.202106116 ◽

2021 ◽

Vol 220 (12) ◽

Author(s):

Delisa E. Clay ◽

Heidi S. Bretscher ◽

Erin A. Jezuit ◽

Korie B. Bush ◽

Donald T. Fox

Keyword(s):

Dna Damage ◽

Dna Polymerase ◽

Chromosome Segregation ◽

Genome Instability ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

End Joining ◽

Strand Breaks ◽

Dna Damage Signaling ◽

Alternative End Joining

Cycling cells must respond to DNA double-strand breaks (DSBs) to avoid genome instability. Missegregation of chromosomes with DSBs during mitosis results in micronuclei, aberrant structures linked to disease. How cells respond to DSBs during mitosis is incompletely understood. We previously showed that Drosophilamelanogaster papillar cells lack DSB checkpoints (as observed in many cancer cells). Here, we show that papillar cells still recruit early acting repair machinery (Mre11 and RPA3) and the Fanconi anemia (FA) protein Fancd2 to DSBs. These proteins persist as foci on DSBs as cells enter mitosis. Repair foci are resolved in a stepwise manner during mitosis. DSB repair kinetics depends on both monoubiquitination of Fancd2 and the alternative end-joining protein DNA polymerase θ. Disruption of either or both of these factors causes micronuclei after DNA damage, which disrupts intestinal organogenesis. This study reveals a mechanism for how cells with inactive DSB checkpoints can respond to DNA damage that persists into mitosis.

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DNA Damage and Repair Mechanisms Triggered by Exposure to Bioflavonoids and Natural Compounds

10.5772/intechopen.95453 ◽

2021 ◽

Author(s):

Donna Goodenow ◽

Kiran Lalwani ◽

Christine Richardson

Keyword(s):

Dna Damage ◽

Cellular Response ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

End Joining ◽

Strand Breaks ◽

Environmental Agents ◽

Repair Mechanisms ◽

Alternative End Joining ◽

Differentiation Status

Eukaryotic cells use homologous recombination (HR), classical end-joining (C-NHEJ), and alternative end-joining (Alt-EJ) to repair DNA double-strand breaks (DSBs). Repair pathway choice is controlled by the activation and activity of pathways specific proteins in eukaryotes. Activity may be regulated by cell cycle stage, tissue type, and differentiation status. Bioflavonoids and other environmental agents such as pesticides have been shown to biochemically act as inhibitors of topoisomerase II (Top2). In cells, bioflavonoids directly lead to DNA double-strand breaks through both Top2-dependent and independent mechanisms, as well as induce DNA damage response (DDR) signaling, and promote alternative end-joining and chromosome alterations. This chapter will present differences in expression and activity of proteins in major DNA repair pathways, findings of Top2 inhibition by bioflavonoids and cellular response, discuss how these compounds trigger alternative end-joining, and conclude with implications for genome instability and human disease.

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No Overt Nucleosome Eviction at Deprotected Telomeres

Molecular and Cellular Biology ◽

10.1128/mcb.01764-07 ◽

2008 ◽

Vol 28 (18) ◽

pp. 5724-5735 ◽

Cited By ~ 28

Author(s):

Peng Wu ◽

Titia de Lange

Keyword(s):

Cellular Response ◽

Double Strand Breaks ◽

Nonhomologous End Joining ◽

Dna Double Strand Breaks ◽

End Joining ◽

Atm Kinase ◽

Strand Breaks ◽

Damage Response ◽

Atr Kinase ◽

Atm Signaling

ABSTRACT Dysfunctional telomeres elicit the canonical DNA damage response, which includes the activation of the ATM or ATR kinase signaling pathways and end processing by nonhomologous end joining (NHEJ) or homologous recombination (HR). The cellular response to DNA double-strand breaks has been proposed to involve chromatin remodeling and nucleosome eviction, but whether dysfunctional telomeres undergo chromatin reorganization is not known. Here, we report on the nucleosomal organization of telomeres that have become deprotected through the deletion of the shelterin components TRF2 or POT1. We found no evidence of changes in the nucleosomal organization of the telomeric chromatin or nucleosome eviction near the telomere terminus. An unaltered chromatin structure was observed at telomeres lacking TRF2, which activate the ATM kinase and are a substrate for NHEJ. Similarly, telomeres lacking POT1a and POT1b, which activate the ATR kinase, showed no overt nucleosome eviction. Finally, telomeres lacking TRF2 and Ku70, which are processed by HR, appeared to maintain their original nucleosomal organization. We conclude that ATM signaling, ATR signaling, NHEJ, and HR at deprotected telomeres can take place in the absence of overt nucleosome eviction.

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