Replication Repair

工作概述：DNA损伤会导致细胞死亡或突变，这两种情况都会对健康造成严重后果。大多数DNA损伤在造成伤害之前就被修复了。经典定义的DNA修复机制是损伤的直接逆转、损伤的切除和受损链的再合成、绕过损伤而不去除损伤的重组过程和诱变翻译合成。在T4噬菌体中发现了第四种但特征不明确的机制。在诱导多种DNA损伤的治疗后，某些DNA复制基因的突变降低了噬菌体的存活，但突变几乎不影响噬菌体的繁殖。这一过程不涉及任何经典机制，由于涉及DNA复制酶，因此被命名为“复制修复”。我们建立了Alberts-Nossal噬菌体T4 8蛋白系统，用于体外复制这两条DNA链，并比较了野生型和突变型酶对受损DNA的处理。突变的DNA结合蛋白对单链DNA的亲和力降低。使用人工复制叉，我们可以观察到模板切换，其中阻断的前导链可以切换到另一子链，以便在阻断后继续合成。在这个实验中，突变蛋白在促进链转换方面有很强的缺陷。

Summary of Work: DNA damage produces cell death or mutation, both of which can have serious health consequences. Most DNA damage is repaired before it can cause harm. The classically defined mechanisms of DNA repair are direct reversal of the damage, excision of the damage followed by resynthesis of the damaged strand, recombinational processes that bypass the damage without removing it, and mutagenic translesion synthesis. A fourth but poorly characterized mechanism was identified in bacteriophage T4. Mutations in certain genes of DNA replication reduce survival after treatments inducing a variety of kinds of DNA damage, but the mutations hardly affect phage reproduction. The process does not involve any of the classical mechanisms and was named "replication repair" because it involves enzymes of DNA replication. We have set up the Alberts-Nossal bacteriophage T4 eight-protein system for replicating both strands of DNA in vitro and have investigated the processing of damaged DNA comparing wild-type and mutant enzymes. A mutant DNA-binding protein exhibits reduced affinity for single-stranded DNA. Using an artificial replication fork, we can observe template switching in which a blocked leading strand can switch to the other daughter strand in order to continue synthesis beyond the block. In this assay, the mutant proteins are strongly defective in promoting strand switching.