Department of Therapeutic Radiology, The Yale Comprehensive Cancer Center, New Haven, CT, USA.
EMBO J. 2010 Jan 20;29(2):410-23. doi: 10.1038/emboj.2009.357. Epub 2009 Dec 17.
We have shown earlier that DNA polymerase beta (Pol beta) localizes to the synaptonemal complex (SC) during Prophase I of meiosis in mice. Pol beta localizes to synapsed axes during zygonema and pachynema, and it associates with the ends of bivalents during late pachynema and diplonema. To test whether these localization patterns reflect a function for Pol beta in recombination and/or synapsis, we used conditional gene targeting to delete the PolB gene from germ cells. We find that Pol beta-deficient spermatocytes are defective in meiotic chromosome synapsis and undergo apoptosis during Prophase I. We also find that SPO11-dependent gammaH2AX persists on meiotic chromatin, indicating that Pol beta is critical for the repair of SPO11-induced double-strand breaks (DSBs). Pol beta-deficient spermatocytes yielded reduced steady-state levels of the SPO11-oligonucleotide complexes that are formed when SPO11 is removed from the ends of DSBs, and cytological experiments revealed that chromosome-associated foci of replication protein A (RPA), RAD51 and DMC1 are less abundant in Pol beta-deficient spermatocyte nuclei. Localization of Pol beta to meiotic chromosomes requires the formation of SPO11-dependent DSBs. Taken together, these findings strongly indicate that Pol beta is required at a very early step in the processing of meiotic DSBs, at or before the removal of SPO11 from DSB ends and the generation of the 3' single-stranded tails necessary for subsequent strand exchange. The chromosome synapsis defects and Prophase I apoptosis of Pol beta-deficient spermatocytes are likely a direct consequence of these recombination defects.
我们之前已经证明,DNA 聚合酶 β(Pol β)在小鼠减数分裂前期 I 中定位于联会复合体(SC)。Pol β 在合线期和粗线期定位于联会轴,在粗线期末期和双线期末期与二价体的末端结合。为了测试这些定位模式是否反映了 Pol β 在重组和/或联会中的功能,我们使用条件基因靶向从生殖细胞中删除 PolB 基因。我们发现,Pol β 缺陷的精母细胞在减数分裂染色体联会中存在缺陷,并在前期 I 中发生凋亡。我们还发现,依赖 SPO11 的 γH2AX 在减数染色质上持续存在,表明 Pol β 对于 SPO11 诱导的双链断裂(DSB)的修复至关重要。Pol β 缺陷的精母细胞产生的 SPO11-寡核苷酸复合物的稳态水平降低,当 SPO11 从 DSB 末端去除时会形成这些复合物,细胞学实验表明,染色体相关的复制蛋白 A(RPA)、RAD51 和 DMC1 焦点在 Pol β 缺陷的精母细胞核中较少。Pol β 向减数分裂染色体的定位需要 SPO11 依赖性 DSB 的形成。综上所述,这些发现强烈表明 Pol β 在减数分裂 DSB 处理的非常早期步骤中是必需的,即在 SPO11 从 DSB 末端去除和生成随后链交换所需的 3'单链尾巴之前或之后。Pol β 缺陷的精母细胞的染色体联会缺陷和前期 I 凋亡很可能是这些重组缺陷的直接后果。