Fig. 4.

ATR ablation does not alter DSB levels but leads to reduction in leptotene recombinase counts. a–d Analysis of SPO11-oligo complexes in P13 testes. SPO11 was immunoprecipitated from whole-testis extracts and SPO11-associated oligos were end-labeled with terminal deoxynucleotidyl transferase and [α-³²P] dCTP, then either separated on SDS-PAGE gels followed by autoradiography (a) and western blotting with anti-SPO11 antibody (b) or digested with proteinase K and resolved on denaturing polyacrylamide sequencing gels (c; background-subtracted lane traces in d). A representative experiment is shown. An additional Atr^fl/+ Cre– control is shown to demonstrate that the Ngn3-Cre transgene does not influence SPO11-oligo levels. In a, b, the bar indicates SPO11-oligo complexes, arrowhead indicates the immunoglobulin heavy chain and asterisk marks non-specific labeling; ND not determined. Each panel shows lanes from the same exposure of a single western blot or autoradiograph, with intervening lanes omitted. For quantitation, SPO11-oligo complex signals were background-subtracted and normalized to Atr^fl/+ Cre– (n = 2) controls. SPO11-oligo quantitation: Atr^fl/+ (1.05 ± 0.07-fold, mean and s.d., n = 2 males), Atr^fl/– (1.17 ± 0.22-fold, n = 4 males), Atm^−/− (14.58 ± 5.24-fold, n = 3 males) and Atr^fl/– Atm^−/− males (11.38 ± 13.15-fold). The reduced SPO11 protein levels in Atm^−/− were previously documented^{40, 79}, but the molecular basis is not understood. e–j Analysis of leptotene focus counts using SYCP3 (green) and early recombination markers (magenta): RPA (e, f), RAD51 (g, h) and DMC1 (i, j) in Atr^fl/+ males (n = 2 males; 27 cells for RPA, 19 cells for RAD51, 14 cells for DMC1), Atr^fl/– males (n = 2 males; 21 cells for RPA, 19 cells for RAD51, 13 cells for DMC1), Atm^−/− males (n = 2 males; 22 cells for RPA, 15 cells for RAD51, 13 cells for DMC1) and Atr^fl/– Atm^−/− males (n = 2 males; 10 cells for RPA, 20 cells for RAD51, 13 cells for DMC1). RPA counts are not significantly different between Atr^fl/– males (mean 249 foci) and Atr^fl/+ males without the Ngn3-Cre transgene (mean 227 foci, n = 2 males, 17 cells), and are thus not influenced by Atr haploinsufficiency. Mean and p values (Mann–Whitney test) indicated. Scale bars 10 µm