Beuning Penny J, Simon Sharotka M, Godoy Veronica G, Jarosz Daniel F, Walker Graham C
Department of Biology, Massachusetts Institute of Technology, Cambridge, USA.
Methods Enzymol. 2006;408:318-40. doi: 10.1016/S0076-6879(06)08020-7.
Members of the Y family of DNA polymerases are specialized to replicate lesion-containing DNA. However, they lack 3'-5' exonuclease activity and have reduced fidelity compared to replicative polymerases when copying undamaged templates, and thus are potentially mutagenic. Y family polymerases must be tightly regulated to prevent aberrant mutations on undamaged DNA while permitting replication only under conditions of DNA damage. These polymerases provide a mechanism of DNA damage tolerance, confer cellular resistance to a variety of DNA-damaging agents, and have been implicated in bacterial persistence. The Y family polymerases are represented in all domains of life. Escherichia coli possesses two members of the Y family, DNA pol IV (DinB) and DNA pol V (UmuD'(2)C), and several regulatory factors, including those encoded by the umuD gene that influence the activity of UmuC. This chapter outlines procedures for in vivo and in vitro analysis of these proteins. Study of the E. coli Y family polymerases and their accessory factors is important for understanding the broad principles of DNA damage tolerance and mechanisms of mutagenesis throughout evolution. Furthermore, study of these enzymes and their role in stress-induced mutagenesis may also give insight into a variety of phenomena, including the growing problem of bacterial antibiotic resistance.
DNA聚合酶Y家族的成员专门负责复制含有损伤的DNA。然而，它们缺乏3'-5'核酸外切酶活性，在复制未受损模板时与复制性聚合酶相比保真度降低，因此具有潜在的致突变性。Y家族聚合酶必须受到严格调控，以防止在未受损DNA上发生异常突变，同时仅在DNA损伤的条件下才允许复制。这些聚合酶提供了一种DNA损伤耐受机制，赋予细胞对多种DNA损伤剂的抗性，并与细菌的持久性有关。Y家族聚合酶在生命的所有领域都有代表。大肠杆菌拥有Y家族的两个成员，DNA聚合酶IV（DinB）和DNA聚合酶V（UmuD'(2)C），以及几个调控因子，包括由umuD基因编码的那些影响UmuC活性的因子。本章概述了对这些蛋白质进行体内和体外分析的程序。对大肠杆菌Y家族聚合酶及其辅助因子的研究对于理解DNA损伤耐受的广泛原理和整个进化过程中的诱变机制非常重要。此外，对这些酶及其在应激诱导诱变中的作用的研究也可能有助于深入了解各种现象，包括日益严重的细菌抗生素耐药性问题。
