Human Rev1 relies on insert-2 to promote selective binding and accurate replication of stabilized G-quadruplex motifs.
Human Rev1 relies on insert-2 to promote selective binding and accurate replication of stabilized G-quadruplex motifs.
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DOI：
10.1093/nar/gkab041
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发表时间：
2021-02-26
影响因子：
14.9
通讯作者：
Eoff RL
中科院分区：
文献类型：
--
作者：
Ketkar A;Smith L;Johnson C;Richey A;Berry M;Hartman JH;Maddukuri L;Reed MR;Gunderson JEC;Leung JWC;Eoff RL
我们先前报道了人Rev 1（hRev 1）以高亲和力结合来自c-MYC启动子的平行链G-四链体（G4）。我们已经将这些结果扩展到包括其他G4基序，发现hRev 1对平行链G4的亲和力比反平行或杂交折叠更强。插入片段-2的αE螺旋中的氨基酸被鉴定为对G4结合重要。将E466和Y 470突变为丙氨酸选择性地扰乱G4结合亲和力。E466 K突变体恢复了野生型G4结合特性。使用正向诱变测定，我们发现与对照序列相比，hRev 1的缺失使G4突变频率增加>200倍。碱基替换和缺失发生在G4基序周围和内部。吡啶抑制素（PDS）加剧了这种效应，因为突变频率比对照增加了>700倍，并且G4位点上游的缺失增加了一倍以上。野生型和E466 K hRev 1部分挽救了G4 DNA（±PDS）的突变复制。E466 A或Y 470 A突变体不能抑制PDS诱导的G4突变频率的增加。这些研究结果的作用插入-2，在脊椎动物中保守的基序，但不是酵母或植物，在Rev 1介导的抑制G4复制过程中的诱变的影响。
We previously reported that human Rev1 (hRev1) bound to a parallel-stranded G-quadruplex (G4) from the c-MYC promoter with high affinity. We have extended those results to include other G4 motifs, finding that hRev1 exhibited stronger affinity for parallel-stranded G4 than either anti-parallel or hybrid folds. Amino acids in the αE helix of insert-2 were identified as being important for G4 binding. Mutating E466 and Y470 to alanine selectively perturbed G4 binding affinity. The E466K mutant restored wild-type G4 binding properties. Using a forward mutagenesis assay, we discovered that loss of hRev1 increased G4 mutation frequency >200-fold compared to the control sequence. Base substitutions and deletions occurred around and within the G4 motif. Pyridostatin (PDS) exacerbated this effect, as the mutation frequency increased >700-fold over control and deletions upstream of the G4 site more than doubled. Mutagenic replication of G4 DNA (±PDS) was partially rescued by wild-type and E466K hRev1. The E466A or Y470A mutants failed to suppress the PDS-induced increase in G4 mutation frequency. These findings have implications for the role of insert-2, a motif conserved in vertebrates but not yeast or plants, in Rev1-mediated suppression of mutagenesis during G4 replication.
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64.8
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