Department of Cancer Research and Molecular Medicine and the FUGE Proteomics Laboratory, Norwegian University of Science and Technology, Trondheim, Norway.
DNA Repair (Amst). 2012 Jan 2;11(1):82-91. doi: 10.1016/j.dnarep.2011.10.026. Epub 2011 Dec 5.
Single-strand DNA binding proteins protect DNA from nucleolytic damage, prevent formation of secondary structures and prevent premature reannealing of DNA in DNA metabolic transactions. In eukaryotes, the nuclear single-strand DNA binding protein RPA is essential for chromosomal DNA replication and transcription and directly participates in several DNA repair processes by binding to and modulating the activity of repair factors. Much less is known about the involvement of the only mitochondrial single-strand binding protein mtSSB in the context of DNA repair. Here we demonstrate that mtSSB impedes excision of uracil and oxidative demethylation of 3meC in single-stranded DNA by UNG1 and ABH1, respectively, whereas excision by NEIL1 was partially inhibited. mtSSB also effectively inhibited nicking of single-stranded DNA by APE1 and ABH1 and partially inhibited the lyase activity of NEIL1. Finally we identified a putative surface motif in mtSSB that may recruit UNG1 to DNA-bound mtSSB. We suggest that the massive amount of mtSSB in mitochondria effectively prevents processing of uracil and other types of damaged bases to avoid introduction of nicks in single-stranded mtDNA formed during replication. Local enrichment of UNG1 at DNA-bound mtSSB may furthermore facilitate rapid access to- and processing of the damage once the dsDNA conformation is restored. This could be of potential biological importance, since mitochondria have no or limited capacity for homologous recombination to process nicks at the replication fork.
单链 DNA 结合蛋白可防止 DNA 受到核酸酶的损伤，防止二级结构的形成，并防止 DNA 代谢过程中 DNA 的过早复性。在真核生物中，核单链 DNA 结合蛋白 RPA 对于染色体 DNA 的复制和转录是必不可少的，并且通过与修复因子结合并调节其活性，直接参与几种 DNA 修复过程。关于唯一的线粒体单链结合蛋白 mtSSB 在 DNA 修复中的参与，人们知之甚少。在这里，我们证明 mtSSB 可分别通过 UNG1 和 ABH1 抑制单链 DNA 中尿嘧啶的切除和 3meC 的氧化去甲基化，而 NEIL1 的切除则部分受到抑制。mtSSB 还能有效地抑制 APE1 和 ABH1 对单链 DNA 的切割，并部分抑制 NEIL1 的裂解酶活性。最后，我们鉴定出 mtSSB 中的一个假定表面模体，该模体可能将 UNG1 募集到与 DNA 结合的 mtSSB 上。我们认为，线粒体中大量的 mtSSB 可有效地防止尿嘧啶和其他类型的受损碱基的处理，以避免在复制过程中形成的单链 mtDNA 中产生缺口。UNG1 在与 DNA 结合的 mtSSB 上的局部富集可能进一步促进 dsDNA 构象恢复后对损伤的快速进入和处理。这可能具有潜在的生物学重要性，因为线粒体几乎没有或没有同源重组的能力来处理复制叉处的缺口。
