Escherichia coli Rep protein and helicase IV

Abstract

Rep protein and helicase IV, two DNA-dependent adenosine 5'-triphosphatases with helicase activity, have been purified from Escherichza coli and characterized. Both enzymes exhibit a distributive interaction with single-stranded DNA as DNA-dependent ATPases in a reaction that is relatively resistant to increasing NaCl concentration and sensitive to the addition of E. coli single-stranded DNA binding protein (SSB). The helicase reaction catalyzed by each protein has been characterized using a direct unwinding assay and partial duplex DNA substrates. Both Rep protein and helicase IV catalyzed the unwinding of a duplex region 71 bp in length. However, unwinding of a 119-bp or 343-bp duplex region was substantially reduced compared to unwinding of the 71-bp substrate. At each concentration of protein examined, the number of base pairs unwound was greatest using the 71-bp substrate, intermediate with the 119-bp substrate and lowest using the 343-bp substrate. The addition of E. coli SSB did not increase the fraction of the 343-nucleotide fragment unwound by Rep protein. However, the addition of SSB did stimulate the unwinding reaction catalyzed by helicase IV approximately twofold. In addition, ionic strength conditions which stabilize duplex DNA (i.e. addition of MgC12 or NaCl), markedly inhibited the helicase reaction catalyzed by either Rep protein or helicase IV while having little effect on the ATPase reaction. Thus, these two enzymes appear to share a common biochemical mechanism for unwinding duplex DNA which can be described as limited unwinding of duplex DNA. Taken together these data suggest that, in vitro, and in the absence of additional proteins, neither Rep protein nor helicase IV catalyzes a processive unwinding reaction.

Genetic Recombination in Bacillus subtilis 168: Effect of Δ helD on DNA Repair and Homologous Recombination

“…It has been shown previously that (i) E. coli helicase IV is a DNA helicase (26), (ii) it shares several biochemical properties with the E. coli helicase II and Rep proteins (27), (iii) double helicase II (uvrD Eco )-helicase IV (helD Eco ) deletion mutants are defective in DNA recombination and repair (20), (iv) B. subtilis possesses two genes homologous to the uvrD Eco -rep Eco tandem, the pcrA and yjcD genes (reference 24; also see above), (v) PcrA, which suppresses the UV sensitivity defect of a uvrD Eco mutant, is an essential helicase of B. subtilis, whereas a yjcD mutant does not exhibit a UV-sensitive phenotype (24). To learn whether the helD gene is involved in recombinational DNA repair, we have constructed a ⌬helD deletion strain.…”

Mutations in theE. coliRep helicase increase the amount of DNA per cell

“…The rep gene of E. coli encodes a leading strand (3′ to 5′ polarity of unwinding) DNA helicase [7–9,21,22]. While Rep is required for the replication of several phage in vitro [23], it is not essential for replication of the chromosome.…”

“…I report here that mbrA4 is a unique allele of rep . Rep is a non‐essential, leading strand (3′ to 5′ polarity) DNA helicase [7–9]. rep(mbrA4) contains three single base mutations: a mutation at −44; an opal stop at position 142; and a glycine to serine change at codon 414 (G414S).…”

The limited strand-separating activity of the UvrAB protein complex and its role in the recognition of DNA damage

“…A length dependence for fragment separation has been seen in distributive helicases, but it is not as marked as we have observed with UvrAB, where an increase in length of only 5–8 nucleotides can prevent release of an oligonucleotide from the duplex almost completely. For example, Rep protein and helicase IV unwind 119‐mers from the duplex regions less effectively than 71‐mers (Yancey‐Wrona et al ., 1992). However, the decrease in activity observed for these helicases is much less dramatic than the decrease in the activity of UvrAB from 28% for a 22‐mer to 2.5% for a 27‐mer (see Figure 1).…”

“…Typically, helicases was also enhanced on the 27-mer (Figure 3, substrate 2), being~8 times higher compared with the release of the separate stretches of duplex DNA ranging from 50 to 25 000 nucleotides long and use the energy of NTP same 27-mer from a substrate without a lesion (3 Ϯ 2% release; three experiments). UvrAB released only 12% of hydrolysis to move unidirectionally along DNA, disrupting the hydrogen bonds linking the two strands (Matson and the 30-mer oligonucleotide (Figure 3, substrate 3), showing a length dependence for the substrates in Figure 3 similar Kaiser-Rogers, 1990;Matson, 1991). The helicase activity to the ssDNA circle and moves along that strand in a 5Ј to 3Ј direction displacing the annealed strand until becoming blocked at the site of damage in the circle.…”