Mammalian BTBD12 (SLX4) Protects against Genomic Instability during Mammalian Spermatogenesis

Abstract

The mammalian ortholog of yeast Slx4, BTBD12, is an ATM substrate that functions as a scaffold for various DNA repair activities. Mutations of human BTBD12 have been reported in a new sub-type of Fanconi anemia patients. Recent studies have implicated the fly and worm orthologs, MUS312 and HIM-18, in the regulation of meiotic crossovers arising from double-strand break (DSB) initiating events and also in genome stability prior to meiosis. Using a Btbd12 mutant mouse, we analyzed the role of BTBD12 in mammalian gametogenesis. BTBD12 localizes to pre-meiotic spermatogonia and to meiotic spermatocytes in wildtype males. Btbd12 mutant mice have less than 15% normal spermatozoa and are subfertile. Loss of BTBD12 during embryogenesis results in impaired primordial germ cell proliferation and increased apoptosis, which reduces the spermatogonial pool in the early postnatal testis. During prophase I, DSBs initiate normally in Btbd12 mutant animals. However, DSB repair is delayed or impeded, resulting in persistent γH2AX and RAD51, and the choice of repair pathway may be altered, resulting in elevated MLH1/MLH3 focus numbers at pachynema. The result is an increase in apoptosis through prophase I and beyond. Unlike yeast Slx4, therefore, BTBD12 appears to function in meiotic prophase I, possibly during the recombination events that lead to the production of crossovers. In line with its expected regulation by ATM kinase, BTBD12 protein is reduced in the testis of Atm−/− males, and Btbd12 mutant mice exhibit increased genomic instability in the form of elevated blood cell micronucleus formation similar to that seen in Atm−/− males. Taken together, these data indicate that BTBD12 functions throughout gametogenesis to maintain genome stability, possibly by co-ordinating repair processes and/or by linking DNA repair events to the cell cycle via ATM.

Resolvase OsGEN1 Mediates DNA Repair by Homologous Recombination

“…Lynn et al, 2007;Guiraldelli et al, 2013) and Class II COs depend on HJ resolvase Mus81-Mms4 (de los Santos et al, 2003;Osman et al, 2003;Hollingsworth and Brill, 2004;Higgins et al, 2008). SLX4 has also been reported to play an important role in Class II CO formation in mice (Holloway et al, 2011).…”

Fan1 deficiency results in DNA interstrand cross-link repair defects, enhanced tissue karyomegaly, and organ dysfunction

“…Fanca-and Fancd2-deficient mice of the 129SV background were obtained from Marcus Grompe (Noll et al 2002;Houghtaling et al 2003). Slx4-and Mus81-deficient mice of the C57/Bl6 background were obtained from Paula Cohen (Dendouga et al 2005;Holloway et al 2011). Genotyping was carried out by PCR on DNA isolated from mouse tail biopsy samples digested overnight in 0.4 mg/ mL Proteinase K supplemented with Direct PCR lysis reagent (yolk sac; Viagenbiotech).…”

RNF4 and PLK1 are required for replication fork collapse in ATR-deficient cells

“…Cells were then cultured for an additional 24 h after 4-OHT washout to afford effective depletion of the ATR protein, at which time APH treatments were initiated. SV40 large T immortalized Slx4 +/+ and Slx4 flox/flox MEFs (Holloway et al 2011) were generously provided by Dr. Paula Cohen, Dr. Robert Weiss, and Dr. Marcus Smolka (Cornell University). Cre-ERT2 was introduced into Slx4 +/+ and Slx4 flox/flox cells through lentivirus transduction, as previously described (Brown and Baltimore 2003).…”