MATERIALS AND METHODS
E. coli strains, plasmid DNAs, and bacteriological techniques.
UV sensitivity and mutation frequency measurements.
Protein purification and in vitro primer extension assays.
RESULTS AND DISCUSSION
Modest overexpression of Pol I partially suppresses the temperature-sensitive growth phenotype of the dnaN159 strain.
The dnaN159 strain is impaired for ssDNA gap repair.
The β159 clamp is impaired for stimulating Pol I and Klenow replication on both a singly primed ssDNA and a nicked dsDNA template in vitro.
Pol I and Pol V compete with each other in the dnaN159 strain.
Evidence for a more prominent role for Pol I in DNA replication in the dnaN159 strain.
Lagging-strand replication is more severely impaired than leading-strand replication in the dnaN159 strain.
Summary and conclusions.
|Straina||dnaN allele||Orientation of lacZ[GGG→ GAG] alleleb||Mutants/106 CFUc|
|lacZ[GGG→ GAG]→ Lac+||Rifr|
|Mutation frequency||Effect (n-fold) (leading/lagging)||Mutation frequency||Effect (n-fold) (leading/lagging)|
|JL100||dnaN+||Leading||0.60 ± 0.13||2.9||4.87 ± 1.77||1.3|
|JL101||dnaN +||Lagging||0.21 ± 0.05||3.74 ± 0.22|
|JL102||dnaN159||Leading||1.40 ± 0.47||0.9||7.31 ± 1.95||1.1|
|JL103||dnaN159||Lagging||1.59 ± 1.29||6.59 ± 1.23|
|Nucleotide positionb||rpoB mutations identified in straina:|
|JL100 (dnaN +)||JL103 (dnaN159)|
|Base substitution||No. of occurrences||Base substitution||No. of occurrences|
|1546||GC→ AT||4||GC→ AT||10|
|1576||GC→ AT||5||CG→ AT||4|
|1586||GC→ AT||1||GC→ AT||4|
|1592||GC→ AT||1||GC→ AT||3|
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