Absence Of DNA-binding Protein Research Articles

Studies of the Silencing of Baculovirus DNA Binding Protein

Baculovirus DNA binding protein (DBP) binds preferentially single-stranded DNA in vitro and colocalizes with viral DNA replication sites. Here, its putative role as viral replication factor has been addressed by RNA interference. Silencing of DBP in Autographa californica multiple nucleopolyhedrovirus-infected cells increased expression of LEF-3, LEF-4, and P35. In contrast, expression of the structural genes coding for P39 and polyhedrin was suppressed while expression of genes coding for P10 and GP64 was unaffected. In the absence of DBP, viral DNA replication sites were formed, indicating replication of viral DNA. Electron microscopy studies, however, revealed a loss of formation of polyhedra and virus envelopment, suggesting that the primary role of DBP is viral formation rather than viral DNA replication.

Spontaneous sharp bending of DNA: role of melting bubbles

The role of centrally located and distributed base pair mismatches (‘melting bubbles’) on localized bending and stiffness of short dsDNA fragments is evaluated using time-dependent fluorescence lifetime measurements. Distributed melting bubbles are found to induce larger bending angles and decreased levels of stiffness in DNA than centrally located ones of comparable overall size. Our results indicate that spontaneous local opening-up of the DNA duplex could facilitate sharp bending of short DNA strands even in the absence of DNA binding proteins. We also find that the occurrence of two closely spaced melting bubbles will generally be favored when a large energetic barrier must be overcome in forming the desired bent DNA structure.

Multimerization of the adenovirus DNA-binding protein is the driving force for ATP-independent DNA unwinding during strand displacement synthesis.

In contrast to other replication systems, adenovirus DNA replication does not require a DNA helicase to unwind the double-stranded template. Elongation is dependent on the adenovirus DNA-binding protein (DBP) which has helix-destabilizing properties. DBP binds cooperatively to single-stranded DNA (ssDNA) in a non-sequence-specific manner. The crystal structure of DBP shows that the protein has a C-terminal extension that hooks on to an adjacent monomer which results in the formation of long protein chains. We show that deletion of this C-terminal arm results in a monomeric protein. The mutant binds with a greatly reduced affinity to ssDNA. The deletion mutant still stimulates initiation of DNA replication like the intact DBP. This shows that a high affinity of DBP for ssDNA is not required for initiation. On a single-stranded template, elongation is also observed in the absence of DBP. Addition of DBP or the deletion mutant has no effect on elongation, although both proteins stimulate initiation on this template. Strand displacement synthesis on a double-stranded template is only observed in the presence of DBP. The mutant, however, does not support elongation on a double-stranded template. The unwinding activity of the mutant is highly reduced compared with intact DBP. These data suggest that protein chain formation by DBP and high affinity binding to the displaced strand drive the ATP-independent unwinding of the template during adenovirus DNA replication.

Unwinding and rewinding of DNA by the RecBC enzyme

Under physiological conditions the initial action of the RecBC enzyme (exonuclease V) on duplex DNA is unwinding of the DNA strands. We have examined by electron microscopy the initial products of this unwinding reaction. When such reactions are carried out in the presence of DNA binding protein, unwinding structures are seen both at the terminus of the duplex DNA and at locations remote from the ends of the DNA molecule. Both terminal and internal unwinding structures proceed along DNA at about 300 nucleotides per second, and the single-stranded loops in both types of structure enlarge at about 100 nucleotides per second. In the internal unwindings DNA must be rewound behind the enzyme at about 200 nucleotides per second. The structures do not occur on supercoiled or nicked circular DNA, indicating that free ends are needed for their formation. In the absence of DNA binding protein only internal unwinding structures are seen, suggesting that the internal structures are formed from the terminal unwindings by base-pairing of their unwound single-strand tails. We present a model which incorporates these structures and is consistent with previous observations on the unwinding and degradative actions of the enzyme. In this model the enzyme travels through duplex DNA by unwinding the DNA ahead of itself and rewinding it behind itself. The internal unwindings produced by the RecBC enzyme could be active in the initial synapsis step in genetic recombination.

Purification of DNA-binding proteins of bacteriophage T3 and their role in in vitro packaging of phage T3 DNA

Concatemers of phage DNA are produced in T3 wild-type-infected cells, but no concatemeric DNA is formed in cells infected with amber mutants of T3 gene 3 (3 −-infected cells) or 6 −-infected cells. Three proteins with molecular weights of 32,000, 31,000, and 16,000 are missing in 3 −-infected cells. The cell extract from 3 −-infected cells (3 −-extract) is defective for in vitro packaging of T3 mature DNA. Two proteins which can complement 3 −-extract for in vitro DNA packaging are purified. These proteins are single-stranded DNA-binding proteins (DBPs) with molecular weights of 32,000 and 31,000, which are identical to two of the three proteins missing in 3 −-infected cells. The DBPs have no endonucleolytic activity. 3 −-Extract also lacks endonucleolytic activity. T3 mature DNA is converted to concatemers by the concerted action of the DBPs and the gene 6 product (gp6). The concatemeric DNA is packaged in vitro in the absence of DBPs and gp6. These results indicate that phage T3 DBPs and gp6 play essential roles in the formation of concatemeric DNA during the T3 infection process.