Differential Rates of NTP Hydrolysis by the Mutant [S69G]RecA Protein: EVIDENCE FOR A COUPLING OF NTP TURNOVER TO DNA STRAND EXCHANGE

Sunil Nayak,Floyd R Bryant

doi:10.1074/jbc.274.37.25979

Abstract

The x-ray crystal structure of the Escherichia coli RecA protein indicates that the phosphate groups of the nucleotide cofactor are bound by a loop whose amino acid sequence ((66)GPESSGKT(73)) corresponds to a consensus phosphate binding loop sequence (GXXXXGK[T/S]) found in many NTP-binding proteins. As part of an investigation of the role of the P-loop in ATP hydrolysis, we prepared a mutant RecA protein in which serine 69 was replaced by a glycine residue. We have found that the [S69G]RecA mutation has a differential effect on the hydrolysis of various nucleoside triphosphates. The [S69G]RecA protein catalyzes the single-stranded DNA-dependent hydrolysis of rATP, ddATP, and dATP with turnover numbers of 10, 20, and 36 min(-1), respectively. The wild type RecA protein, in contrast, hydrolyzes each of these nucleoside triphosphates with similar turnover numbers of 20-24 min(-1). Significantly, the [S69G]RecA protein promotes strand exchange with all three nucleoside triphosphates, and the rate of strand exchange is directly proportional to the rate of hydrolysis of each of the nucleotide cofactors. These findings with the [S69G]RecA protein provide support for the existence of a mechanistic coupling between NTP hydrolysis and DNA strand exchange.

Highlights

The x-ray crystal structure of the Escherichia coli RecA protein indicates that the phosphate groups of the nucleotide cofactor are bound by a loop whose amino acid sequence (66GPESSGKT73) corresponds to a consensus phosphate binding loop sequence (GXXXXGK[T/S]) found in many NTP-binding proteins
In order to quantify the rates of the wild type and [S69G]RecA protein-promoted strand exchange reactions, the intensity of the agarose gel bands corresponding to the linear dsDNA substrate, the strand exchange intermediates, and the circular dsDNA strand exchange product at each time point in the reactions shown in Fig. 3A was measured by scanning densitometry
Cox and co-workers (7) showed that the RecA protein-promoted ATP hydrolysis and strand exchange reactions have a similar dependence on temperature over the range of 25– 45 °C, a finding that is consistent with a coupling of strand exchange to ATP hydrolysis

Summary

EVIDENCE FOR A COUPLING OF NTP TURNOVER TO DNA STRAND EXCHANGE*

The x-ray crystal structure of the Escherichia coli RecA protein indicates that the phosphate groups of the nucleotide cofactor are bound by a loop whose amino acid sequence (66GPESSGKT73) corresponds to a consensus phosphate binding loop sequence (GXXXXGK[T/S]) found in many NTP-binding proteins. The most extensively investigated DNA pairing activity is the ATPdependent three-strand exchange reaction in which a circular ssDNA1 molecule and a homologous linear dsDNA molecule are recombined to yield a nicked circular dsDNA molecule and a linear ssDNA molecule The x-ray crystal structure of the RecA protein indicates that the phosphate groups of the nucleotide cofactor, ATP, are bound by a loop consisting of amino acids 66 through 73 (2) The sequence of this loop (66GPESSGKT73) corresponds to a variation of the well known phosphate binding loop (P-loop) consensus sequence (GXXXXGK[T/S]) found in many NTP-binding proteins (3). The biochemical properties of the [S69G]RecA protein provide new insight into the coupling of NTP hydrolysis and DNA strand exchange and are described in this report

EXPERIMENTAL PROCEDURES

RESULTS

Wild type

Wild type ATP

DISCUSSION