Fig 1.
Purification of His-tagged RecBCD complex and mutant enzymes by Ni-NTA agarose column chromatography.
(A) Steps involved in purification of wild-type RecBCD and mutant proteins. (B) SDS-PAGE analysis of purified RecBCD protein fractions of wild-type and mutants. Purified protein fractions were stained either with coomassie brilliant blue (B) or with silver nitrate (C). Three protein bands of expected size corresponding to RecB, RecC and RecD are visible on the gel. A low molecular protein (~60 kDa) observed on silver nitrate strained gel was identified as GroEL, a HSP-60 family chaperonin.
Fig 2.
The dsDNA-dependent ATPase activity of wild-type and mutant RecBCD enzymes at different temperatures.
The assays were carried out by TLC method as described in Methods. (A) A graph showing the concentration dependent ATP hydrolysis by RecBCD (WT) and mutant enzymes at 22°C. The inset in B shows a blow-up of the same data of mutant enzymes using an expanded y-scale. (B) Representative of TLC plates showing the dsDNA-dependent ATP hydrolysis by wild-type and mutant RecBCD enzymes at 37°, 22° and 4°C. (C) Arrhenius plot of ATPase activity of wildtype and mutant enzymes at 37°, 22° and 4°C. The logarithmic rates vs inverse of the absolute temperature is plotted and the linear slope of each enzyme is indicated. There is no apparent difference in slope (EA/R, where EA is activation energy and R is the gas constant) is observed despite reduced ATP hydrolysis in ATPase mutants of RecBCD enzyme. The ATP hydrolysis data presented are the results obtained from three independent experiments.
Table 1.
ATPase activity of wild type and mutant RecBCD enzymes.
Fig 3.
Effects of magnesium and ATP on the unwinding and degradation by wild-type RecBCD enzyme of P. syringae.
The DNA unwinding and degradation assays were performed with [5′-32P] labeled NdeI linearized pBR322 plasmid DNA in the presence of different concentrations of magnesium and ATP, as described in Methods; (A) the reaction mixture contained fixed amount of Mg-acetate (2 mM) and the amount ATP was varied (0 to 10 mM) as indicated. (B) The reaction mixture contained the fixed amount of ATP (2 mM) and concentration of Mg-acetate was varied (0 to 10 mM) as indicated. The reactions were performed for 5 min and analyzed on a 1% agarose gel containing 1X TBE buffer at a 25–30 Volts for 15 hrs. Agarose gels were dried, exposed to phosphor imaging plates and quantified using Phosphor Imager (Fuji-3000). The data were analyzed using image gauge software. The lanes C1 and C2 contain [5′-32P] labeled dsDNA and the heat denatured 5′-32P labeled dsDNA respectively as a control. A [5′-32P] labeled discrete DNA bands of smaller than full-length ssDNA of pBR322 were also noticed in the lanes 5–10 of panel B.
Fig 4.
DNA unwinding and degradation of NdeI digested linear dsDNA of pBR322 by RecBCDPs (WT) enzyme at 22° and 4°C.
The DNA unwinding and degradation at 22° and 4°C were carried out as described in Methods. (A) The DNA unwinding reactions contained 5 mM ATP and 2 mM Mg++ (limiting magnesium condition) (B) The DNA degradation reactions contained 2 mM ATP and 6 mM Mg++ (excess magnesium condition). The reactions were initiated by adding ATP, and stopped at the indicated times by adding stop-buffer. The lanes C1 and C2 contain [5′-32P] labeled NdeI linearised double-stranded and the heat-denatured ssDNA of pBR322 as control. The discrete ssDNA fragments of pBR322 produced by the nuclease activity of RecBCD enzyme are also indicated.
Table 2.
DNA unwinding and degradation activities of the wild type and mutant RecBCD enzymes at 22 and 4°C.
Fig 5.
DNA unwinding and degradation of NdeI linearized dsDNA of pBR322 by mutant RecBCDPs enzymes at 22° and 4°C.
(A) DNA unwinding and degradation by RecBK28QCD enzyme. The DNA unwinding and degradation at 22° and 4°C were carried out as described in Methods. Note that this enzyme shows DNA unwinding (ssDNA production), but no detectable DNA degradation at 22°C, and none were detectable at 4°C. (B) DNA unwinding and degradation by RecBCDK229Q enzyme. The RecBCDK229Q enzyme has apparently retained both the DNA unwinding and degradation properties. But, interestingly, the discrete DNA bands are absent (C) DNA unwinding by nuclease-deficient RecBD1118ACD enzyme under limiting and excess magnesium conditions at 22 and 4°C. RecBD1118ACD enzyme is unable to degrade DNA at both 22 and 4°C and notably, the DNA unwinding seems to be faster in excess-magnesium reaction condition (2 mM ATP:6 mM Mg++) compared to limiting magnesium reaction condition (5 mM ATP:2 mM Mg++). Each reaction mixtures contained 0.5 nM of enzyme and 10 μM (nucleotides) linear [5′-32P] labeled pBR322 dsDNA. The lanes C1 and C2 contain [5′-32P] labeled NdeI linearized double-stranded and the heat-denatured ssDNA of pBR322 respectively.
Fig 6.
Identification of Chi sequence using PCR amplified pBR322 fragment (3.6 kb) containing internal deletions as a DNA substrate.
(A) Top panel; Schematic representation of NdeI linearized pBR322 plasmid DNA. The locations of OROPI and OPROPII primers used in PCR amplification of 3.6 kb fragments are indicated. Bottom panel; DNA degradation reactions performed using the PCR amplified 3.6 kb DNA as substrate, having either bottom strand labeled (left panel) or top strand labeled (middle panel) or both strand labeled (right panel). The lane C contains [5′-32P] labeled heat-denatured ssDNA as a control. Notably, the top strand labeling of DNA substrate resulted in appearance of discrete DNA fragments. (B) Deletion of 400–450 bp of pBR322 (pBR322(Δ400–450)) resulted in disappearance of DNA fragments. But, it is clearly visible when intact 3.6 kb pBR322 was used as a substrate, suggesting the presence of putative ChiPs sequence in this region. (C) Further deletion of 401–419; 421–439 bo regions; and, (D) 421–429; 431–439; 441–449 bp regions of pBR322 shows that the DNA fragments are indeed from the 431–439 bp region of the pBR322 DNA sequence. (E) A schematic representation of 3.6 kb region of pBR322 and deleted regions within, are shown in the left panel. Right panel shows the presence (+) or absence (-) of intense protected band (Chi-like fragments), when these constructs are used as assay substrates.
Fig 7.
(A) DNA degradation of modified fragment of pBR322 by RecBCDPs enzyme. Apart from one 8-mer ChiPs sequence at 431th position of pBR322, there are two 7-mer similar sequences present in the plasmid pBR322. One such similar sequence at 964th position (5′ GCTGGCGT 3′) was mutated to make it an 8-mer sequence (5′ GCTGGCGC 3′). This substrate (pBR322T971C) that has two Chi sequences in the same orientation when used as a substrate, yielded two DNA fragments with high intensity. (B) The DNA degradation pattern of XbaI digested linear dsDNA of pBKS plasmid by RecBCDPs enzyme. ChiPs are inserted into pBluescript vector (pBKS) by site directed insertion. XbaI linearized pBKS vector or pBKS with ChiPs was 5′-end labeled with 32P has been used for assays. The discrete ssDNA of expected size was produced when pBKS (ChiPs) was used as a substrate, whereas it is absent, when native pBKS was used.
Table 3.
Chi sequences identified in different bacteria.
Fig 8.
Role of RecBCD dependent DNA repair pathway in rescue of low temperature induced replication forks arrest.
(i) A chromosome replicating bi-directionally, (ii) encounters low temperature-induced chromosomal lesion or blockage, (iii), causing replication fork arrest and fork reversal (RFR). RFR is suppressed by linearized chromosomal DNA degradation by RecBCD enzyme and resetting of replication fork. (iv) RFR is stabilized by RuvAB complex and, (v) further, resolved by RuvC leading to chromosomal linearization. Linearized chromosome is processed by the nuclease activity of RecBCD enzyme alone or by RecBCD-dependent homologous recombination process. The defective motors activity of RecBCD enzyme leads to chromosomal fragmentation and cell death at the low temperature.