The role of ADP-ribosylation in regulating DNA double-strand break repair.

ADP-ribosylation is the post translational modification of proteins catalysed by ADP-ribosyltransferases (ARTs). ADP-ribosylation has been implicated in a wide variety of cellular processes including cell growth and differentiation, apoptosis and transcriptional regulation. Perhaps the best characterised role, however, is in DNA repair and genome stability where ADP-ribosylation promotes resolution of DNA single strand breaks. Although ADP-ribosylation also occurs at DNA double strand breaks (DSBs), which ARTs catalyse this reaction and the molecular basis of how this modification regulates their repair remains a matter of debate. Here we review recent advances in our understanding of how ADP-ribosylation regulates DSB repair. Specifically, we highlight studies using the genetic model organism Dictyostelium, in addition to vertebrate cells that identify a third ART that accelerates DSB repair by non-homologous end-joining through promoting the interaction of repair factors with DNA lesions. The implications of these data with regards to how ADP-ribosylation regulates DNA repair and genome stability are discussed.

PARP regulates nonhomologous end joining through retention of Ku at double-strand breaks.

Poly adenosine diphosphate (ADP)-ribosylation (PARylation) by poly ADP-ribose (PAR) polymerases (PARPs) is an early response to DNA double-strand breaks (DSBs). In this paper, we exploit Dictyostelium discoideum to uncover a novel role for PARylation in regulating nonhomologous end joining (NHEJ). PARylation occurred at single-strand breaks, and two PARPs, Adprt1b and Adprt2, were required for resistance to this kind of DNA damage. In contrast, although Adprt1b was dispensable for PARylation at DSBs, Adprt1a and, to a lesser extent, Adprt2 were required for this event. Disruption of adprt2 had a subtle impact on the ability of cells to perform NHEJ. However, disruption of adprt1a decreased the ability of cells to perform end joining with a concomitant increase in homologous recombination. PAR-dependent regulation of NHEJ was achieved through promoting recruitment and/or retention of Ku at DSBs. Furthermore, a PAR interaction motif in Ku70 was required for this regulation and efficient NHEJ. These data illustrate that PARylation at DSBs promotes NHEJ through recruitment or retention of repair factors at sites of DNA damage.

DNA double-strand break repair pathway choice in Dictyostelium.

DNA double-strand breaks (DSBs) can be repaired by homologous recombination (HR) or non-homologous end joining (NHEJ). The mechanisms that govern whether a DSB is repaired by NHEJ or HR remain unclear. Here, we characterise DSB repair in the amoeba Dictyostelium. HR is the principal pathway responsible for resistance to DSBs during vegetative cell growth, a stage of the life cycle when cells are predominantly in G2. However, we illustrate that restriction-enzyme-mediated integration of DNA into the Dictyostelium genome is possible during this stage of the life cycle and that this is mediated by an active NHEJ pathway. We illustrate that Dclre1, a protein with similarity to the vertebrate NHEJ factor Artemis, is required for NHEJ independently of DNA termini complexity. Although vegetative dclre1(-) cells are not radiosensitive, they exhibit delayed DSB repair, further supporting a role for NHEJ during this stage of the life cycle. By contrast, cells lacking the Ku80 component of the Ku heterodimer that binds DNA ends to facilitate NHEJ exhibit no such defect and deletion of ku80 suppresses the DSB repair defect of dclre1(-) cells through increasing HR efficiency. These data illustrate a functional NHEJ pathway in vegetative Dictyostelium and the importance of Ku in regulating DSB repair choice during this phase of the life cycle.

The Ino80 chromatin-remodeling complex restores chromatin structure during UV DNA damage repair.

Chromatin structure is modulated during deoxyribonucleic acid excision repair, but how this is achieved is unclear. Loss of the yeast Ino80 chromatin-remodeling complex (Ino80-C) moderately sensitizes cells to ultraviolet (UV) light. In this paper, we show that INO80 acts in the same genetic pathway as nucleotide excision repair (NER) and that the Ino80-C contributes to efficient UV photoproduct removal in a region of high nucleosome occupancy. Moreover, Ino80 interacts with the early NER damage recognition complex Rad4-Rad23 and is recruited to chromatin by Rad4 in a UV damage-dependent manner. Using a modified chromatin immunoprecipitation assay, we find that chromatin disruption during UV lesion repair is normal, whereas the restoration of nucleosome structure is defective in ino80 mutant cells. Collectively, our work suggests that Ino80 is recruited to sites of UV lesion repair through interactions with the NER apparatus and is required for the restoration of chromatin structure after repair.