HELIX-DESTABILIZING ACTIVITY OF PHI-29 SINGLE-STRANDED-DNA BINDING-PROTEIN - EFFECT ON THE ELONGATION RATE DURING STRAND DISPLACEMENT DNA-REPLICATION
HELIX-DESTABILIZING ACTIVITY OF PHI-29 SINGLE-STRANDED-DNA BINDING-PROTEIN - EFFECT ON THE ELONGATION RATE DURING STRAND DISPLACEMENT DNA-REPLICATION
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DOI：
10.1006/jmbi.1995.0570
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发表时间：
1995-11-03
影响因子：
5.6
通讯作者：
SALAS, M
中科院分区：
文献类型：
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作者：
SOENGAS, MS;GUTIERREZ, C;SALAS, M
噬菌体Phi 29的单链DNA（ssDNA）结合蛋白（SSB）是病毒DNA复制所需的病毒编码蛋白之一。我们发现Phi 29 SSB具有螺旋去稳定活性，因为它去除Phi 29复制中间体中ssDNA的二级结构，如电子显微镜所示，并取代退火至M13 ssDNA的寡核苷酸。为了研究SSB依赖性刺激Phi 29 DNA复制的机制，我们表征了Phi 29 SSB的螺旋去稳定活性，并通过Phi 29 DNA聚合酶测量了其对DNA延伸率的影响，该聚合酶不需要辅助解旋酶。使用复制反应，其中需要链置换，（Phi 29 DNA复制）或不（将引发的M13 ssDNA转化为双链DNA（dsDNA））使我们发现（1）链置换DNA复制受到降低温度或增加盐浓度的影响，由于在Phi 29模板上的DNA延伸速率比在引发的M13 ssDNA上慢三到四倍，（2）在这些条件下，添加Phi 29 SSB在Phi 29 DNA复制期间不同程度地刺激DNA延伸速率，而它对引发的M13 ssDNA复制具有边际效应，（3）Phi 29 SSB使Phi 29 DNA聚合酶链置换突变体的Phi 29 DNA延伸率增加4 - 6倍，达到野生型酶的50%左右。螺旋不稳定的性质的Phi 29 SSB的条件下，DNA开放受损的影响进行了讨论。(C)1995年学术出版社
The single-stranded DNA (ssDNA)-binding protein (SSB) of bacteriophage Phi 29 is one of the virus-encoded proteins required for viral DNA replication. We have found that Phi 29 SSB has helix-destabilizing activity since it removes secondary structure of the ssDNA in Phi 29 replicative intermediates, as revealed by electron microscopy, and displaces oligonucleotides annealed to M13 ssDNA. To investigate the mechanism of the SSB-dependent stimulation of Phi 29 DNA replication we have characterized the helix-destabilizing activity of Phi 29 SSB and measured its effect on the DNA elongation rate by Phi 29 DNA polymerase, which does not require an accessory helicase. The use of replication reactions where strand displacement is either required (Phi 29 DNA replication) or not (conversion of primed M13 ssDNA into double-stranded DNA (dsDNA)) has allowed us to find that (1) strand displacement DNA replication was affected by lowering the temperature or by increasing the salt concentration, since the DNA elongation rate on the Phi 29 template was three to fourfold slower than on primed M13 ssDNA, (2) under those conditions, addition of Phi 29 SSB stimulated to different extents the DNA elongation rate during Phi 29 DNA replication, whereas it had a marginal effect on primed M13 ssDNA replication, and (3) Phi 29 SSB increased four to sixfold the Phi 29 DNA elongation rate by Phi 29 DNA polymerase strand displacement mutants, reaching similar to 50% the rate of the wild-type enzyme. The implications of the helix-destabilizing properties of the Phi 29 SSB under conditions in which DNA opening is impaired are discussed. (C) 1995 Academic Press Limited