Isolation of an altered form of DNA polymerase I from Escherichia coli cells induced for recA/lexA functions.

Abstract

A novel form of DNA polymerase I (deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase, DNA nucleotidyltransferase, EC 2.7.7.7) activity has been isolated from Escherichia coli cells that had been activated for expression of the DNA damage-inducible genes. Induction was by treatment ofnormal cells or cells carrying the #pr-51 and tif-1 mutations with nalidixic acid. This activity, DNA polymerase I*, seems to be a form ofDNA polymerase I because it is insensitive toN-ethylmaleimide, is inhibited by antibody to DNA polymerase I, and does not appear in a polAl strain. DNA polymerase I* activity sediments through sucrose gradients as a broad peak with s2 ,0 = 6.6-10.5, compared with an s2o,. = 4.8-5.5 for DNA polymerase I. The fidelity during polymerization reactions of DNA polymerase I* is relatively low with a variety of synthetic templates and deoxynucleoside triphosphates, although the enzyme appears to have a normal level of 3'->5' exonuclease. Polymerase I* has properties that might implicate it in some form of mutagenic DNA repair.Mutations induced in Escherichia coli by UV radiation or by a variety of chemicals are believed to often be the consequence of an error-prone DNA repair pathway controlled, along with other SOS functions, by the recA and lexA genes (1-3). This pathway is evidently induced by agents that block DNA replication either by interfering with replicative enzymes or by modifying bases so as to affect base recognition in the DNA template. Blocks due to base damage are presumably mediated by constraints that regulate replicative fidelity by ensuring the synthesis only of properly base-paired DNA. Models have been proposed for overcoming such replicative blocks that predict the transient appearance of error-prone forms of DNA polymerase that can polymerize random nucleotides opposite template damage (2,4). Because such hypotheses are consistent with much of the available data on mutagenic DNA repair (5), we have sought to identify these predicted forms of DNA polymerase. We report here the observation ofan error-prone form of DNA polymerase I that is associated with the induction of recA/lexA functions in E. coli. MATERIALS AND METHODSBacterial Strains. Extracts were prepared from E. coli K-12 strains DM1187 (tif-1, spr-51, lexA3, sftAll, his4, strA31) (6), AB1157 (argE3, his4, leu-6, proA2, thr-1, ara-14, galK2, lacYl, mtll, xyl5, thi-l, tsx-33, supE44, sup-37, str-31) (7), and P3478 (polAl, thyA36), a derivative of W3110 (5).Materials. Synthetic polymers and unlabeled dNTPs were from P-L Biochemicals, radioactive nucleotides were from Amersham, and electrophoretically homogeneous DNA polymerase I was a gift from Arthur Kornberg, Stanford University, or Lawrence Loeb, University ofWashington. Antibody against DNA polymerase I was provided by I. R. Lehman, Stanford University. Polymin-P was from Miles; agarose (type II) was from Sigma; DNA-agarose was prepared by the method of Schaller et aL (8), except that DNA had been denatured by heating to 950C.Sucrose Gradient Sedimenta...

Treatment with DNA-damaging agents increases expression of polA`-'lacZ gene fusions in Escherichia coli K-12

“…However, although the notion has arisen that Pol I expression is constitutive (Kornberg 1980), a growing body of evidence suggests that polA gene expression may be regulated by several unknown mechanisms, which still remain to be elucidated. These mechanisms seem to be related to DNA repair (Ahmad and van Sluis 1987;Lackey et al 1982Lackey et al , 1985, growth rate regulation (Polayes et al 1988a,b) and cell metabolism (Hickey and Hirshfield 1990;Shapiro 1992). Our results provide further evidence for the existence of regulatory mechanisms governing polA gene expression, and are the first data reporting increased polA'-'lacZ expression following the action of DNA-damaging agents that interfere with DNA replication.…”

“…Spot 42 RNA is a moderately abundant, stable RNA species encoded by the spf gene located immediately downstream of polA . Finally, an altered form of Pol I (Pol I*) exhibiting substantially decreased fidelity has been isolated from SOS-induced cells (Lackey et al 1982(Lackey et al , 1985. However, since a growing body of evidence argues for DNA polymerase III as the major SOS-responsive polymerase (Bridges et al 1976, Brotkorne-Lannoye et al 1987Hagensee et al 1987;Rajagolapan et al 1992), the true role of Pol I* in SOS repair and mutagenesis remains to be elucidated.…”

Functional recA, lexA, umuD, umuC, polA, and polB genes are not required for the Escherichia coli UVM response

“…The existence of polA null mutants suggests that other polymerases are able to compensate for the loss of pol I activity. In SOS-induced cells, pol I was reported to acquire a new form suggestive of a role in error-prone replication (33,34); however, UV mutability appears to be unaffected in polA strains (4). pol II, encoded by the SOS gene polB (dinA) (7,14,26,55), can bypass noninstructive lesions efficiently in vitro (7) but is nevertheless not required for bypass of such lesions in vivo (31).…”

Translesion synthesis is the main component of SOS repair in bacteriophage lambda DNA

“…5). In addition, the appearance of a novel form of E. coli DNA polymerase I, Poll*, after induction of the SOS system has been reported (23,24). This relatively low-fidelity polymerase could also account for the capacity of the cell to replicate damaged template.…”