Biology subjects

Wood, R. D.

Publications and source records attributed to Wood, R. D..

4 recordsLinked to original sources

bioRxiv2024

Repeat expansion in a Fragile X model is independent of double strand break repair mediated by Pol θ, Rad52, Rad54l or Rad54b

Microsatellite instability is responsible for the human Repeat Expansion Disorders. The mutation responsible differs from classical cancer-associated microsatellite instability (MSI) in that it requires the mismatch repair proteins that normally protect against MSI. LIG4, an enzyme essential for non-homologous end-joining (NHEJ), the major pathway for double-strand break repair (DSBR) in mammalian cells, protects against expansion in mouse models. Thus, NHEJ may compete with the expansion pathway for access to a common intermediate. This raises the possibility that expansion involves an NHEJ-independent form of DSBR. Pol {theta}, a polymerase involved in the theta-mediated end joining (TMEJ) DSBR pathway, has been proposed to play a role in repeat expansion. Here we examine the effect of the loss of Pol {theta} on expansion in FXD mouse embryonic stem cells (mESCs), along with the effects of mutations in Rad52, Rad54l and Rad54b, genes important for multiple DSBR pathways. None of these mutations significantly affected repeat expansion. These observations put major constraints on what pathways are likely to drive expansion. Together with our previous demonstration of the protective effect of nucleases like EXO1 and FAN1, and the importance of Pol {beta}, they suggest a plausible model for late steps in the expansion process.

genetics↗

bioRxiv2020

Deficiency in Translesion DNA Polymerase ζ Induces an Innate Immune Response

DNA polymerase pol {zeta} is regarded as a specialized DNA polymerase for bypass of DNA lesions. In mammalian cells, pol {zeta} also contributes to genomic stability during normal DNA replication. Disruption of Rev3l (the catalytic subunit of pol {zeta}) is toxic to cells and mice, with increased constitutive chromosome damage, including micronuclei. As the cellular manifestations of this genomic stress have remained unexplored, we measured genome-wide transcriptional changes by RNA-seq in pol {zeta}-defective cells. Expression of 1117 transcripts was altered by 4-fold or more in Rev3l knockout mouse embryonic fibroblasts (MEFs), with a pattern showing an induction of an innate immune response. We validated the increased expression of known interferon-stimulated genes (ISG) at the mRNA and protein levels. We found that the cGAS-STING axis, which senses cytosolic DNA, drives ISG expression in Rev3l knockout MEFs. These results reveal a new genome protective function of pol {zeta} and indicate that inhibition of pol {zeta} may be therapeutically useful by simultaneously increasing sensitivity to genotoxins and inducing a cytotoxic innate immune response.

cell biology↗

bioRxiv2019

Mechanistic Basis for Microhomology Identification and Genome Scarring by Polymerase Theta

DNA Polymerase Theta mediates an end joining pathway (TMEJ) that repairs chromosome breaks. It requires resection of broken ends to generate long, 3 single stranded DNA tails, annealing of complementary sequence segments (microhomologies) in these tails, followed by microhomology-primed synthesis sufficient to resolve broken ends. The means by which microhomologies are identified is thus a critical step in this pathway, but is not understood. Here we show microhomologies are identified by a scanning mechanism initiated from the 3 terminus and favoring bi-directional progression into flanking DNA, typically to a maximum of 15 nucleotides into each flank. Polymerase theta is frequently insufficiently processive to complete repair of breaks in microhomology-poor, AT-rich regions. Aborted synthesis leads to one or more additional rounds of microhomology search, annealing, and synthesis; this promotes complete repair in part because earlier rounds of synthesis generate microhomologies de novo that are sufficiently long that synthesis is more processive. Aborted rounds of synthesis are evident in characteristic genomic scars as insertions of 3-30 bp of sequence that is identical to flanking DNA ("templated" insertions). Templated insertions are present at higher levels in breast cancer genomes from patients with germline BRCA1/2 mutations, consistent with an addiction to TMEJ in these cancers. Our work thus describes the mechanism for microhomology identification, and shows both how it mitigates limitations implicit in the microhomology requirement, and generates distinctive genomic scars associated with pathogenic genome instability.

molecular biology↗

bioRxiv2018

FAM35A associates with REV7 and modulates DNA damage responses of normal and BRCA1-defective cells

In order to exploit the specific vulnerabilities of tumors, it is urgent to identify the basis of associated defects in genome maintenance. One unsolved problem is the mechanism of inhibition of processing of DNA double-strand break repair by REV7 and its influence on DNA repair pathways. We searched for REV7-associated proteins in human cells and found FAM35A, a protein of previously unknown function. By analyzing the FAM35A sequence we discovered that FAM35A has an unstructured N-terminal region and a C-terminal region harboring three OB-fold domains similar to single-stranded binding protein RPA. Knockdown of FAM35A caused sensitivity to DNA damaging agents, and FAM35A re-localized in damaged cell nuclei. In a BRCA1 mutant cell line, however, depletion of FAM35A increased resistance to camptothecin, suggesting that FAM35A participates in processing of DNA ends to allow more efficient DNA repair. Moreover, we found FAM35A absent in one widely used BRCA1-mutant cancer cell line (HCC1937) with anomalous resistance to PARP inhibitors. A survey of FAM35A alterations in cancer revealed that the gene is altered at the highest frequency in prostate cancers (up to 13%) and significantly less expressed in metastatic cases. The results reveal a new DNA repair factor with promise as a therapeutically relevant cancer marker.

molecular biology↗