SAW1 is increasingly required to recruit Rad10 as SSA flap-length increases from 20 to 50 bases in single-strand annealing in S. cerevisiae.
SAW1 is increasingly required to recruit Rad10 as SSA flap-length increases from 20 to 50 bases in single-strand annealing in S. cerevisiae.
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DOI：
10.1016/j.bbrep.2021.101125
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发表时间：
2021-12
影响因子：
2.7
通讯作者：
Fischhaber PL
中科院分区：
文献类型：
--
作者：
Odango RJ;Camberos J;Fregoso FE;Fischhaber PL
SAW 1是Rad 1-Rad 10核酸酶所必需的，用于有效去除在S.酿酒酵母，单链退火（SSA）和合成依赖性链退火（SDSA）。Saw 1在体外显示出随着瓣长度从0到40个脱氧核苷酸（nt）变化而对瓣DNA表现出增加的亲和力，当瓣短于10 nt时几乎没有观察到结合。因此，我们先前的体内荧光显微镜研究表明，SAW 1是不需要的招聘Rad 10-YFP的DNA双链断裂（DSB）时，皮瓣是10 nt，但它是需要的，当皮瓣是1500 nt的细胞周期的G1期。我们很好奇，我们是否也会观察到增加的要求SAW 1的Rad 10招聘在体内皮瓣从20至50 nt，在体外显示。在这项研究中，我们利用SSA底物，产生20，30，和50 nt皮瓣在体内的荧光显微镜测定，并确定SAW 1变得越来越必要的SSA开始在约1020 nt，并完全需要在1050 nt。定量PCR实验证实了这些结果表明，修复产物的形成减少，在没有SAW 1的皮瓣长度增加。用含有荧光标记的Saw 1（Saw 1-CFP）的菌株进行的实验表明，Saw 1与SSA病灶处的Rad 10一起定位，并且在DSBs处含有Rad 10的病灶中约有一半不含Saw 1。Saw 1-CFP的共定位模式是一致的，无论基板的瓣长度，并且在细胞周期的所有阶段大致相似。总之，这些数据表明，Saw 1变得越来越重要的Rad 1-Rad 10招聘和SSA修复在20-50 nt皮瓣范围内，和Saw 1是存在于修复位点，即使不需要，并可能离开修复位点之前Rad 1-Rad 10。随着DNA瓣的增加，对Saw 1募集Rad 1-Rad 10的依赖性增加。导致依赖性增加的瓣长度范围为20-50个脱氧核苷酸。Saw 1被发现位于单链退火灶处，即使不需要募集Rad 1-Rad 10。Saw 1仅在约一半的含有Rad 1-Rad 10的单链退火灶中被发现。
SAW1 is required by the Rad1-Rad10 nuclease for efficient removal of 3′ non-homologous DNA ends (flaps) formed as intermediates during two modes of double-strand break repair in S. cerevisiae, single-strand annealing (SSA) and synthesis-dependent strand annealing (SDSA). Saw1 was shown in vitro to exhibit increasing affinity for flap DNAs as flap lengths varied from 0 to 40 deoxynucleotides (nt) with almost no binding observed when flaps were shorter than 10 nt. Accordingly, our prior in vivo fluorescence microscopy investigation showed that SAW1 was not required for recruitment of Rad10-YFP to DNA double-strand breaks (DSBs) when flaps were ∼10 nt, but it was required when flaps were ∼500 nt in G1 phase of the cell cycle. We were curious whether we would also observe an increased requirement of SAW1 for Rad10 recruitment in vivo as flaps varied from ∼20 to 50 nt, as was shown in vitro. In this investigation, we utilized SSA substrates that generate 20, 30, and 50 nt flaps in vivo in fluorescence microscopy assays and determined that SAW1 becomes increasingly necessary for SSA starting at about ∼20 nt and is completely required at ∼50 nt. Quantitative PCR experiments corroborate these results by demonstrating that repair product formation decreases in the absence of SAW1 as flap length increases. Experiments with strains containing fluorescently labeled Saw1 (Saw1-CFP) show that Saw1 localizes with Rad10 at SSA foci and that about half of the foci containing Rad10 at DSBs do not contain Saw1. Colocalization patterns of Saw1-CFP are consistent regardless of the flap length of the substrate and are roughly similar in all phases of the cell cycle. Together, these data show that Saw1 becomes increasingly important for Rad1-Rad10 recruitment and SSA repair in the ∼20–50 nt flap range, and Saw1 is present at repair sites even when not required and may depart the repair site ahead of Rad1-Rad10. There is an increasing dependence on Saw1 to recruit Rad1-Rad10 as DNA flaps increase The flap length range causing the increasing dependence is 20–50 deoxynucleotides Saw1 is found at single-strand annealing foci even when not required to recruit Rad1-Rad10 Saw1 is found in only about half of the single-strand annealing foci containing Rad1-Rad10
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DOI：
10.1073/pnas.95.5.2390
发表时间：
1998-03-03
影响因子：
11.1
作者：
Strout, MP;Marcucci, G;Caligiuri, MA
通讯作者：
Caligiuri, MA
DOI：
10.1016/j.bbrc.2014.02.048
发表时间：
2014-03-14
影响因子：
3.1
作者：
Diamante, Graciel;Claire Phan;Fischhaber, Paula L.
通讯作者：
Fischhaber, Paula L.
影响因子：
5.3
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Paques, F;Haber, JE
通讯作者：
Haber, JE
影响因子：
21.3
作者：
Lisby, M;Mortensen, UH;Rothstein, R
通讯作者：
Rothstein, R
影响因子：
16
作者：
Li, Fuyang;Dong, Junchao;Lee, Sang Eun
通讯作者：
Lee, Sang Eun