PMID:10559166
| Citation |
McLenigan, MP, Kulaeva, OI, Ennis, DG, Levine, AS and Woodgate, R (1999) The bacteriophage P1 HumD protein is a functional homolog of the prokaryotic UmuD'-like proteins and facilitates SOS mutagenesis in Escherichia coli. J. Bacteriol. 181:7005-13 |
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| Abstract |
The Escherichia coli umuD and umuC genes comprise an operon and encode proteins that are involved in the mutagenic bypass of normally replication-inhibiting DNA lesions. UmuD is, however, unable to function in this process until it undergoes a RecA-mediated cleavage reaction to generate UmuD'. Many homologs of umuDC have now been identified. Most are located on bacterial chromosomes or on broad-host-range R plasmids. One such putative homolog, humD (homolog of umuD) is, however, found on the bacteriophage P1 genome. Interestingly, humD differs from other umuD homologs in that it encodes a protein similar in size to the posttranslationally generated UmuD' protein and not UmuD, nor is it in an operon with a cognate umuC partner. To determine if HumD is, in fact, a bona fide homolog of the prokaryotic UmuD'-like mutagenesis proteins, we have analyzed the ability of HumD to complement UmuD' functions in vivo as well as examined HumD's physical properties in vitro. When expressed from a high-copy-number plasmid, HumD restored cellular mutagenesis and increased UV survival to normally nonmutable recA430 lexA(Def) and UV-sensitive DeltaumuDC recA718 lexA(Def) strains, respectively. Complementing activity was reduced when HumD was expressed from a low-copy-number plasmid, but this observation is explained by immunoanalysis which indicates that HumD is normally poorly expressed in vivo. In vitro analysis revealed that like UmuD', HumD forms a stable dimer in solution and is able to interact with E. coli UmuC and RecA nucleoprotein filaments. We conclude, therefore, that bacteriophage P1 HumD is a functional homolog of the UmuD'-like proteins, and we speculate as to the reasons why P1 might require the activity of such a protein in vivo. |
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| Keywords |
Amino Acid Sequence; Bacterial Proteins/chemistry; Bacterial Proteins/genetics; Bacterial Proteins/physiology; Bacteriophage P1/genetics; Bacteriophage P1/radiation effects; DNA-Directed DNA Polymerase; Escherichia coli/genetics; Escherichia coli/radiation effects; Escherichia coli Proteins; Genetic Complementation Test; Molecular Sequence Data; Mutagenesis; Promoter Regions, Genetic; Rec A Recombinases/metabolism; SOS Response (Genetics); Sequence Alignment; Ultraviolet Rays; Viral Proteins/chemistry; Viral Proteins/genetics; Viral Proteins/isolation & purification; Viral Proteins/physiology |
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