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Genetic mapping and DNA sequence analysis of mutations in the polA gene of Escherichia coli.

Abstract：

:DNA polymerase I of Escherichia coli provides an excellent model for the study of template-directed enzymatic synthesis of DNA because it is a single subunit enzyme, it can be obtained in large quantities and the three-dimensional structure of the polymerizing domain (the Klenow fragment) has recently been determined (Ollis et al., 1985). One approach to assigning functions to particular portions of the structure is to correlate the altered enzymatic behavior of mutant forms of DNA polymerase I with the change in the primary sequence of the protein. Towards this end we have developed a rapid procedure for mapping any polA mutation to a region no larger than 300 base-pairs within the polA gene. Two series of polA deletion mutants with defined end-points were constructed in vitro and cloned into bacteriophage lambda. These phages can then be used to map precisely E. coli polA mutants. Twelve polA- alleles have been mapped in this way and for nine of them the nature of the mutational change has been determined by DNA sequence analysis. Two of the mutations, polA5 and polA6, which affect the enzyme-DNA interaction, provide evidence for the location of the DNA binding region on the polymerase three-dimensional structure.

journal_name

J Mol Biol

journal_title

Journal of molecular biology

authors

Joyce CM,Fujii DM,Laks HS,Hughes CM,Grindley ND

doi

10.1016/0022-2836(85)90105-6

subject

Has Abstract

pub_date

1985-11-20 00:00:00

pages

283-93

issue

2

eissn

0022-2836

issn

1089-8638

pii

0022-2836(85)90105-6

journal_volume

186

pub_type

杂志文章

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