The Escherichia coli UVM response is accompanied by an SOS-independent error-prone DNA replication activity demonstrable in vitro.
The Escherichia coli UVM response is accompanied by an SOS-independent error-prone DNA replication activity demonstrable in vitro.
复制标题
大肠杆菌 UVM 反应伴随着体外可证明的不依赖于 SOS 的易错 DNA 复制活性。
DOI：
10.1046/j.1365-2958.2000.02136.x
复制
发表时间：
2000
影响因子：
3.6
通讯作者：
Humayun,MZ
中科院分区：
文献类型：
--
作者：
AlMamun,AA;Yadava,RS;Ren,L;Humayun,MZ
UVM是一种SOS非依赖性诱导反应，其特征在于转染至经DNA损伤剂预处理的大肠杆菌细胞中的M13单链DNA上携带的位点特异性3，N4-乙烯胞嘧啶（εC）残基的突变升高。通过构造和使用E.大肠杆菌AM 124（polA polB umuDC dinB lexA 1 [Ind-]），我们在这里表明，UVM反应表现在SOS诱导缺陷的细胞中，以及所有四种“非复制型”DNA聚合酶，即DNA聚合酶I（polA），II（polB），IV（dinB）和V（umuDC）。这些结果证实，UVM代表了一种新的，以前未识别的细胞对DNA损伤剂的反应。为了解决UVM反应是否伴随着易错的DNA复制活性的问题，我们应用了一种新开发的体外复制测定法与体外突变分析系统相结合。在该试验中，在细胞提取物和核苷酸前体存在下，在密切模拟M13体内复制的条件下，带有位点特异性病变的环状M13单链DNA转化为环状双链复制型DNA。新合成的（负）DNA链通过连接介导的聚合酶链反应（LM-PCR）进行选择性扩增，然后进行多重序列分析以确定突变的频率和特异性。未诱导的E细胞提取物复制带有位点特异性εC损伤的DNA。coliAM 124细胞导致约13%的突变频率。在UVM诱导的AM 124细胞的细胞提取物中，突变频率升高了5倍（至58%），C → A突变占主导地位，C → T突变，特异性与体内的突变相似。 这些结果与先前报道的数据一起表明，UVM反应是通过诱导瞬时易错DNA复制活性介导的，DNA聚合酶III的修饰或先前未鉴定的DNA聚合酶的表达可能是UVM表型的原因。
UVM is an SOS‐independent inducible response characterized by elevated mutagenesis at a site‐specific 3, N4‐ethenocytosine (εC) residue borne on M13 single‐stranded DNA transfected intoEscherichia colicells pretreated with DNA‐damaging agents. By constructing and usingE. colistrain AM124 (polA polB umuDC dinB lexA1[Ind–]), we show here that the UVM response is manifested in cells deficient for SOS induction, as well as for all four of the ‘non‐replicative’ DNA polymerases, namely DNA polymerase I (polA), II (polB), IV (dinB) and V (umuDC). These results confirm that UVM represents a novel, previously unidentified cellular response to DNA‐damaging agents. To address the question as to whether the UVM response is accompanied by an error‐prone DNA replication activity, we applied a newly developedin vitroreplication assay coupled to anin vitromutation analysis system. In the assay, circular M13 single‐stranded DNA bearing a site‐specific lesion is converted to circular double‐stranded replicative‐form DNA in the presence of cell extracts and nucleotide precursors under conditions that closely mimic M13 replicationin vivo. The newly synthesized (minus) DNA strand is selectively amplified by ligation‐mediated polymerase chain reaction (LM‐PCR), followed by a multiplex sequence analysis to determine the frequency and specificity of mutations. Replication of DNA bearing a site‐specific εC lesion by cell extracts from uninducedE. coliAM124 cells results in a mutation frequency of about 13%. Mutation frequency is elevated fivefold (to 58%) in cell extracts from UVM‐induced AM124 cells, with C → A mutations predominating over C → T mutations, a specificity similar to that observedin vivo. These results, together with previously reported data, suggest that the UVM response is mediated through the induction of a transient error‐prone DNA replication activity and that a modification of DNA polymerase III or the expression of a previously unidentified DNA polymerase may account for the UVM phenotype.