Linking DNA polymerase theta structure and function in health and disease

Beagan, Kelly; McVey, Mitch

doi:10.1007/s00018-015-2078-9

Cited by 40 publications

(45 citation statements)

References 89 publications

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“…Human Pol θ was shown to interact with Orc2 and Orc4 and thus can localize to replication origins (12). It additionally was suggested that Pol θ is important for the temporal regulation of origin firing, although its exact role remains unclear (12). In contrast, our results show that the timing of origin firing is maintained in lig4; mus308, albeit reduced, and mus308 alone has no effect on the level of origin firing during amplification.…”

Section: How Might Various Repair Mechanisms Affect Replication Forkmentioning

confidence: 45%

“…This overexpression may confer an advantage to cancer cells and alleviate replication stress by promoting DSB repair at sites of replication fork collapse. We therefore investigated whether Pol θ, encoded by mutagen-sensitive 308 (mus308) in Drosophila (12), is involved in DSB repair during rereplication in Drosophila follicle cells and whether its loss might impair the production of proteins involved in eggshell development. Indeed, we found that null mutants in mus308 exhibit a sporadic eggshell defect ( Fig.…”

Section: Absence Of Pol θ Perturbs Chorion Gene Transcription and Eggmentioning

confidence: 99%

Section: How Might Various Repair Mechanisms Affect Replication Forkmentioning

confidence: 99%

“…Microhomology-mediated end joining (MMEJ), an alternative mechanism of end-joining repair that is frequently used in the absence of NHEJ, was also shown to be used for DSB repair during rereplication (6,11). MMEJ requires limited resection to expose single-stranded DNA ends that anneal at small (1-10 bp) microhomologous sequences (12). DNA polymerase θ (Pol θ) mediates the annealing of microhomologies and subsequent fill-in synthesis required during MMEJ in many organisms (12).…”

mentioning

confidence: 99%

“…MMEJ requires limited resection to expose single-stranded DNA ends that anneal at small (1-10 bp) microhomologous sequences (12). DNA polymerase θ (Pol θ) mediates the annealing of microhomologies and subsequent fill-in synthesis required during MMEJ in many organisms (12).…”

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confidence: 99%

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Multiple mechanisms contribute to double-strand break repair at rereplication forks in Drosophila follicle cells

Alexander

Beagan

Orr‐Weaver

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Rereplication generates double-strand breaks (DSBs) at sites of fork collisions and causes genomic damage, including repeat instability and chromosomal aberrations. However, the primary mechanism used to repair rereplication DSBs varies across different experimental systems. In Drosophila follicle cells, developmentally regulated rereplication is used to amplify six genomic regions, two of which contain genes encoding eggshell proteins. We have exploited this system to test the roles of several DSB repair pathways during rereplication, using fork progression as a readout for DSB repair efficiency. Here we show that a null mutation in the microhomology-mediated end-joining (MMEJ) component, polymerase θ/mutagen-sensitive 308 (mus308), exhibits a sporadic thin eggshell phenotype and reduced chorion gene expression. Unlike other thin eggshell mutants, mus308 displays normal origin firing but reduced fork progression at two regions of rereplication. We also find that MMEJ compensates for loss of nonhomologous end joining to repair rereplication DSBs in a site-specific manner. Conversely, we show that fork progression is enhanced in the absence of both Drosophila Rad51 homologs, spindle-A and spindle-B, revealing homologous recombination is active and actually impairs fork movement during follicle cell rereplication. These results demonstrate that several DSB repair pathways are used during rereplication in the follicle cells and their contribution to productive fork progression is influenced by genomic position and repair pathway competition. Furthermore, our findings illustrate that specific rereplication DSB repair pathways can have major effects on cellular physiology, dependent upon genomic context. oogenesis | break-induced replication | DNA repair | NHEJ | fork collision

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Section: How Might Various Repair Mechanisms Affect Replication Forkmentioning

confidence: 45%

Section: Absence Of Pol θ Perturbs Chorion Gene Transcription and Eggmentioning

confidence: 99%

Section: How Might Various Repair Mechanisms Affect Replication Forkmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Multiple mechanisms contribute to double-strand break repair at rereplication forks in Drosophila follicle cells

Alexander

Beagan

Orr‐Weaver

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Moonlighting at replication forks – a new life for homologous recombination proteins BRCA1, BRCA2 and RAD51

et al. 2017

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Edited by Wilhelm JustCoordination between DNA replication and DNA repair ensures maintenance of genome integrity, which is lost in cancer cells. Emerging evidence has linked homologous recombination (HR) proteins RAD51, BRCA1 and BRCA2 to the stability of nascent DNA. This function appears to be distinct from double-strand break (DSB) repair and is in part due to the prevention of MRE11-mediated degradation of nascent DNA at stalled forks. The role of RAD51 in fork protection resembles the activity described for its prokaryotic orthologue RecA, which prevents nuclease-mediated degradation of DNA and promotes replication fork restart in cells challenged by DNA-damaging agents. Here, we examine the mechanistic aspects of HR-mediated fork protection, addressing the crosstalk between HR and replication proteins.Keywords: DNA recombination; DNA replication; genome stability BRCA1, BRCA2, RAD51 and the RAD51 paralogs family, which consists of five proteins (RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3) in mammalian cells, are required to repair DNA damage by homologous recombination (HR). Mutations in most of these genes predispose to cancer, indicating an important role of DNA damage repair in preventing cell transformation. Intriguingly, complete loss of function of most of HR proteins is incompatible with life in vertebrate organisms [1,2,3]. These features together with their ability to form foci in unperturbed and challenged S-phase nuclei indicate a role for HR proteins in chromosomal DNA replication even in unchallenged conditions [4,5].The mechanisms underlying the function of DNA repair proteins in unchallenged chromosomal DNA replication are poorly understood. This is in part due to the fact that many of the genes involved in DNA metabolism are essential for cell viability, which complicate their study, especially in higher eukaryotes [1,2,3]. The reasons why HR genes are essential for cell viability in higher eukaryotes are unclear. One explanation might be that they have a specific and direct role in the replication of complex eukaryotic genomes. Alternatively, complex genomes might be more vulnerable to spontaneous DNA damage, which might irreversibly halt replication progression inducing chromosomes breakage. HR factors might be therefore required to repair the damage and to complete whole genome duplication. Here we review the links between HR proteins and the DNA replication machinery, some of which appear to be conserved between prokaryotes and eukaryotes.

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Expression and Structural Analyses of Human DNA Polymerase θ (POLQ)

Malaby

Martin

Wood

et al. 2017

Methods in Enzymology

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DNA polymerase theta (pol θ) is an evolutionarily conserved protein encoded by the POLQ gene in mammalian genomes. Pol θ is the defining enzyme for a pathway of DSB repair termed “alternative end-joining” (altEJ) or “theta-mediated end-joining”. This pathway contributes significantly to the radiation resistance of mammalian cells. It also modulates accuracy in repair of breaks that occur at stalled DNA replication forks, during diversification steps of the mammalian immune system, during repair of CRISPR-Cas9, and in many DNA integration events. Pol θ is a potentially important clinical target, particularly for cancers deficient in other break repair strategies. The enzyme is uniquely able to mediate joining of single-stranded 3′ ends. Because of these unusual biochemical properties and its therapeutic importance, it is essential to study structures of pol θ bound to DNA. However, challenges for expression and purification are presented by the large size of pol θ (2,590 residues in humans) and unusual juxtaposition of domains (a helicase-like domain and distinct DNA polymerase, separated by a region predicted to be largely disordered). Here we summarize work on the expression and purification of the full-length protein, and then focus on the design, expression and purification of an active C-terminal polymerase fragment. The generation of this active construct was non-trivial and time-consuming. Almost all published biochemical work to date has been performed with this domain fragment. Strategies to obtain and improve crystals of a ternary pol θ complex (enzyme:DNA:nucleotide) are also presented, along with key elements of the structure.

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Linking DNA polymerase theta structure and function in health and disease

Cited by 40 publications

References 89 publications

Multiple mechanisms contribute to double-strand break repair at rereplication forks in Drosophila follicle cells

Multiple mechanisms contribute to double-strand break repair at rereplication forks in Drosophila follicle cells

Moonlighting at replication forks – a new life for homologous recombination proteins BRCA1, BRCA2 and RAD51

Expression and Structural Analyses of Human DNA Polymerase θ (POLQ)

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