DNA REPLICATION, REPAIR, AND MUTAGENESIS IN EUKARYOTIC AND PROKARYOTIC CELLS

DNA损伤在大肠杆菌中诱导的突变发生在瞬间，作为 全球SOS响应。这一进程的一个关键参与者是UMUD 与UMUC和激活的RecA(RecA*)一起使 DNA聚合酶III全酶在化学和紫外线诱导下的复制 损伤。我们的X射线晶体分析表明，除了 形成分子二聚体，氨基末端和羧基末端尾巴 从球状的贝塔结构延伸到联想和生产 结晶的细丝。这些细丝是生物所需的 活性：氨基末端的缺失不会影响 蛋白质形成分子二聚体，但确实严重损害了 UmuD‘与RecA-DNA细丝相互作用并参与的能力 在突变过程中。 由于许多DNA修复过程在结构和/或功能上 在原核生物和真核生物之间保守，我们研究了 RAD30，一个与酿酒酵母DNA修复相关的未知基因 对大肠杆菌UMUC、dinB和酿酒酵母Rev1基因的紫外线抗性 和紫外线诱变。我们的发现表明RAD30参与了 在依赖于RAD6和RAD18的新的无错误修复途径中，但是 独立于Rev1、Rev3、Rev7和RAD5。 我们之前在两个INCL/M质粒上发现了umu互补基因， R471a和R446b。对这些基因的分子分析表明，它们是 与其他成员相比，与MUAB在结构和功能上的关联度更高 像umu一样的家族。我们在MUAB(R471a)上发现了一个5‘插入片段 似乎是一种新的逆转录元件，编码一种假定的反转 转录酶(RT)；该RT与II组编码的RT相关 内含子，但它被嵌入在一个类似倒退的上下文中。这一观察结果 提示R471a中的类黏蛋白AB基因位于 R-质粒可能曾经是(或仍然是)一种可移动的基因 元素。这一观察结果可能解释了类umu的分布。 R-质粒和细菌染色体上的基因。 在修复紫外线诱导的灵长类细胞DNA损伤的工作中，我们有 重点研究了UV-DDB，一个由p127和p48亚基组成的蛋白质复合体 它与6-4个嘧啶二聚体(PD)特异结合。此装订 着色性干皮病(XP)E组未检测到活性；修复 可通过微量注射UV-DDB在XP-E细胞中恢复。但是，在 与所有其他XP蛋白不同的是，UV-DDB不能促进 体外重组修复系统(裸DNA底物)。我们现在有了 发现UV-DDB复合体的另一个成员是RPA，一个已知的 损伤识别蛋白。由于体内需要UV-DDB，但不需要 在体外DNA修复中，我们假设UV-DDB可能识别并结合 6-4个PD并改变周围的染色质结构以允许进入 “修理工”。

DNA damage-induced mutagenesis in E. coli occurs transiently as part of the global SOS response. A key participant in this process is the UmuD' protein which, together with UmuC and activated RecA (RecA*), enables the DNA polymerase III holoenzyme to replicate across chemical and UV-induced lesions. Our x-ray crystallographic analysis shows that in addition to forming molecular dimers, the amino-terminal and carboxyl-terminal tails of UmuD' extend from a globular beta structure to associate and produce crystallized filaments. These filaments are required for biological activity: Deletion of the amino terminus does not affect the ability of the protein to form molecular dimers but does severely compromise the ability of UmuD' to interact with a RecA-DNA filament and to participate in mutagenesis. Since many DNA repair processes are structurally and/or functionally conserved between prokaryotes and eukaryotes, we investigated the role of RAD30, a previously uncharacterized S. cerevisiae DNA repair gene related to the E. coli umuC, dinB, and S. cerevisiae REV1 genes, in UV resistance and UV-induced mutagenesis. Our findings suggest that RAD30 participates in a novel error-free repair pathway dependent on RAD6 and RAD18, but independent of REV1, REV3, REV7 and RAD5. We previously identified umu-complementing genes on two incL/M plasmids, R471a and R446b. Molecular analysis of these genes reveals that they are more structurally and functionally related to mucAB than to other members of the umu-like family. We found an insert 5' to mucAB(R471a) that appears to be a novel retroelement encoding a putative reverse transcriptase (RT); this RT is related to the RTs encoded by group II introns but it is embedded in a retron-like context. This observation suggests that the mucAB-like gene in R471a is located within a region of the R-plasmid that perhaps was once (or still is) a mobile genetic element. This observation might explain the distribution of umu-like genes on R-plasmids and bacterial chromosomes. In work on the repair of UV-induced DNA damage in primate cells, we have focused on UV-DDB, a protein complex comprising p127 and p48 subunits that binds specifically to 6-4 pyrimidine dimers (PD). This binding activity is not detectable in xeroderma pigmentosum (XP) Group E; repair can be restored in XP-E cells by microinjection of UV-DDB. However, in contrast to all other XP proteins, UV-DDB does not promote repair in an in vitro reconstituted repair system (naked DNA substrate). We have now found that another member of the UV-DDB complex is RPA, a known damage-recognition protein. Since UV-DDB is required for in vivo but not in vitro DNA repair, we hypothesize that UV-DDB may recognize and bind to 6-4 PDs and alter the surrounding chromatin structure to permit access of the "repairosome".