Competition between Replicative and Translesion Polymerases during Homologous Recombination Repair in Drosophila

In metazoans, the mechanism by which DNA is synthesized during homologous recombination repair of double-strand breaks is poorly understood. Specifically, the identities of the polymerase(s) that carry out repair synthesis and how they are recruited to repair sites are unclear. Here, we have investigated the roles of several different polymerases during homologous recombination repair in Drosophila melanogaster. Using a gap repair assay, we found that homologous recombination is impaired in Drosophila lacking DNA polymerase zeta and, to a lesser extent, polymerase eta. In addition, the Pol32 protein, part of the polymerase delta complex, is needed for repair requiring extensive synthesis. Loss of Rev1, which interacts with multiple translesion polymerases, results in increased synthesis during gap repair. Together, our findings support a model in which translesion polymerases and the polymerase delta complex compete during homologous recombination repair. In addition, they establish Rev1 as a crucial factor that regulates the extent of repair synthesis

Pilot Study of the Delivery of Microcollimated Pars Plana External Beam Radiation in Porcine Eyes: 270-Day Analysis

Objective. To determine the dose response and toxicity threshold of micro-collimated X-rays delivered to porcine maculae by a stereotactic radiosurgical system after 270 days. Methods. Twelve eyes of six Yucatan mini-swine were randomized to receive up to 90 Gy to the retina, using an office-based trans-pars plana delivery system. To determine the safety profile of this radiation delivery, ophthalmic examination, fundus photography, fluorescein angiography (FA), and spectral domain optical coherence tomography (SD-OCT) were obtained at multiple time points up to 270 days post treatment. Results. No abnormalities were noted on external examination. Cataracts were noted in 4 of 12 eyes. Dose and time-dependent changes were noted on fundus examination, FA, ICG and SD-OCT. No significant abnormalities were seen in the control, 16 Gy or 24 Gy groups using any modality. Histopathology revealed a dose response effect with no discernable lesions in the 16 Gy group. Conclusion. The X-ray delivery system precisely targets the porcine retina in vivo with little effect on surrounding structures. No ophthalmic or intracranial adverse effects were noted at clinically relevant doses at 270 days following radiation delivery

<i>pol32</i> mutants are sensitive to multiple DNA damaging agents.

<p>(A) A null allele (<i>L2</i>) of <i>POL32</i> (<i>CG3975</i>) was created through imprecise excision of a <i>P</i> element (EY15283) located in the 3′ untranslated region (UTR) of the <i>POL32</i> gene. White box indicates the <i>POL32</i> open reading frame; shaded regions, the UTRs; numbers indicate nucleotide position from start of transcription. (B) <i>pol32</i> mutants are sensitive to ionizing radiation (IR). Percent survival was calculated as the percentage of homozygote eclosion relative to an untreated control. (C) <i>pol32</i> mutants are sensitive to methyl methanesulfanate (MMS) and nitrogen mustard (HN2), but not camptothecin (CPT). Error bars represent the standard deviation for at least three trials.</p

<i>pol32</i> mutants are impaired in DNA synthesis during HR repair.

<p>(A) The <i>P{w^a}</i> site-specific repair assay. Expression of transposase in males possessing <i>P{w^a}</i> (i) results in a 14 kilobase gap (ii) relative to an uncut sister chromatid. Full HR requires synthesis of the <i>white</i> gene and <i>copia</i> long terminal repeats (LTRs), followed by annealing at the LTRs (iii). Aborted HR results when end-joining repair occurs prior to synthesis of the entire <i>white</i> gene. Amount of repair synthesis in aborted HR repair events can be estimated by PCR (iv). (B) <i>pol32</i> mutants are significantly impaired in full HR repair relative to wildtype. Wildtype n = 55; <i>pol32</i> n = 120. Error bars represent standard errors. *<i>P</i><0.05, Mann-Whitney test. (C) Repair synthesis is decreased in <i>pol32</i> mutants. Each bar represents the percentage of events with at least the indicated amount of synthesis. Right end: wildtype n = 55; <i>pol32</i> n = 151. Left end: wildtype n = 55 <i>pol32</i> n = 66. *<i>P</i><0.05, Fisher's exact test.</p

Model for polymerase action at a DSB.

<p>Multiple polymerases compete for access to D-loops. Following formation of a double-strand gap, Rev1 binds at the break site(s), recruits pol zeta, and blocks access of other polymerases. Initial synthesis is carried out by pol zeta, which readily dissociates. Repair can then be completed by end joining or another polymerase can bind and reinitiate synthesis. Binding of pol delta and its processivity subunit Pol32 to the D-loop results in processive synthesis and promotes repair of large gaps. Other polymerases, including pol eta, can act in backup roles. Elimination of Rev1 or multiple TLS polymerases increases the probability of pol delta recruitment leading to increased repair synthesis.</p