dsDNA Unwinding Research Articles

Specific defects in double-stranded DNA unwinding and homologous pairing of a mutant RecA protein

The DNA molecules bound to RecA filaments are extended 1.5-fold relative to B-form DNA. This extended DNA structure may be important in the recognition of homology between single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA). In this study, we show that the K286N mutation specifically impaired the dsDNA unwinding and homologous pairing activities of RecA, without an apparent effect on dsDNA binding itself. In contrast, the R243Q mutation caused defective dsDNA unwinding, due to the defective dsDNA binding of the C-terminal domain of RecA. These results provide new evidence that dsDNA unwinding is essential to homology recognition between ssDNA and dsDNA during homologous pairing.

Protein-Protein Interactions of the Primase Subunits p58 and p48 with Simian Virus 40 T Antigen Are Required for Efficient Primer Synthesis in a Cell-free System

DNA polymerase alpha-primase (pol-prim, consisting of p180-p68-p58-p48), and primase p58-p48 (prim(2)) synthesize short RNA primers on single-stranded DNA. In the SV40 DNA replication system, only pol-prim is able to start leading strand DNA replication that needs unwinding of double-stranded (ds) DNA prior to primer synthesis. At high concentrations, pol-prim and prim(2) indistinguishably reduce the unwinding of dsDNA by SV40 T antigen (Tag). RNA primer synthesis on ssDNA in the presence of replication protein A (RPA) and Tag has served as a model system to study the initiation of Okazaki fragments on the lagging strand in vitro. On ssDNA, Tag stimulates whereas RPA inhibits the initiation reaction of both enzymes. Tag reverses and even overcompensates the inhibition of primase by RPA. Physical binding of Tag to the primase subunits and RPA, respectively, is required for these activities. Each subunit of the primase complex, p58 and p48, performs physical contacts with Tag and RPA independently of p180 and p68. Using surface plasmon resonance, the dissociation constants of the Tag/pol-prim and Tag/primase interactions were 1.2 x 10(-8) m and 1.3 x 10(-8) m, respectively.

C-terminal truncated Escherichia coli RecA protein RecA5327 has enhanced binding affinities to single- and double-stranded DNAs

RecA5327 is a truncated RecA protein that is lacking 25 amino acid residues from the C-terminal end. The expression of RecA5327 protein in the cell resulted in the constitutive induction of SOS functions without damage to the DNA. Purified RecA5327 protein effectively promoted the LexA repressor cleavage reaction and ATP hydrolysis at a lower concentration of single-stranded DNA than that required for wild-type RecA protein. A DNA binding study showed that RecA5327 has about ten times higher affinity for single-stranded DNA than does the wild-type RecA protein. Moreover RecA5327 protein binds stably to double-stranded (ds) DNA in conditions where the wild-type RecA protein could not bind. The binding of RecA5327 protein to dsDNA was associated with the unwinding of dsDNA, suggesting that RecA5327 binds to dsDNA in the same manner as does the wild-type protein. The fact that RecA5327 does not bind stoichiometrically but forms short filaments on dsDNA suggests that it nucleates to dsDNA much more frequently than does the wild-type protein. The role of the 25 C-terminal residues, in the regulation of RecA binding to DNA, is discussed.

Diabetes-Related Changes in Chromatin Structure of Brain, Liver, and Intestinal Epithelium

To determine whether diabetes alters chromatin structure in vivo, fluorometric analysis of alkali-induced DNA unwinding was carried out in various tissues of streptozocin-induced diabetic rats and genetically obese diabetic (db/db) mice. When zero-order kinetics were used to analyze the data, the percentage of double-stranded DNA (%dsDNA) unwinding in brain, liver, and intestinal epithelium of diabetic rats maintained for 4 wk was significantly reduced compared with vehicle-injected control rats (%dsDNA 0.37 +/- 0.05 vs. 0.73 +/- 0.02 for brain, 0.59 +/- 0.1 vs. 0.84 +/- 0.02 for liver, and 0.58 +/- 0.07 vs. 0.90 +/- 0.13 for intestinal epithelium). Insulin treatment of diabetic rats normalized the rate of DNA unwinding in liver (0.82 +/- 0.09 %dsDNA/min) and intestinal epithelium (1.05 +/- 0.09 %dsDNA/min), but the increase in the unwinding rate of brain DNA (0.51 +/- 0.06 %dsDNA/min) did not achieve control values. Similarly, alkali-induced DNA unwinding was significantly slower in brain and liver of db/db mice compared with homozygote controls. When first-order kinetics were used to analyze the data, fractional rate constants of DNA unwinding in brain and liver of diabetic rats or mice were significantly smaller than observed in nondiabetic control animals. The fractional rate constant of DNA unwinding in intestinal epithelium was not altered with diabetes. We conclude that chronic uncontrolled hyperglycemia can alter chromatin structure in vivo.

Non-specific "pairing" of DNA molecules by recombination enzyme of Bacillus subtilis.

ATP-dependent deoxyribonuclease(s) (ATP-DNase) is involved in the genetic recombination process in Escherichia coli1–3, Diplococcus pneumoniae4 and Bacillus subtilis5 and may also play an important part in DNA repair6–8, DNA replication9 and cell growth10. ATP-dependent or ATP-stimulated DNases have also been isolated and characterised in other microorganisms11–14. We reported the purification and properties of this enzyme in B. subtilis15,16 and noted that at the 600-fold purification level, the enzyme requires ATP for the hydrolysis of double stranded (ds) DNA, but not for single stranded (ss) DNA16. Since the activities on ds and ssDNA appear to reside in the same enzyme, we suggested the possibility that ATP consumption is somehow linked to the active unwinding of dsDNA before the DNase can hydrolyse phosphodiester bonds16. Recently, Friedman and Smith24 have also proposed an active role of ATP in dsDNA unwinding by Haemophilus influenzae enzyme, which is based on the structural feature of intermediate product DNA. To examine this possibility further, we tried to visualize the unwound portion of DNA and the DNA-enzyme complex by electron microscopy. Although the visual evidence is still ambiguous, an unexpected observation has been made. We report here the unusual property of B. subtilis ATP-DNase which apparently brings DNA molecules into close proximity or causes non-specific ‘pairing’ of dsDNA molecules.