ATP-independent DNA unwinding by the adenovirus single-stranded DNA binding protein requires a flexible DNA binding loop.
Journal - Journal of molecular biology (ENGLAND )
The adenovirus DNA binding protein (DBP) binds cooperatively to single-stranded (ss) DNA and stimulates both initiation and elongation of DNA replication. DBP forms protein filaments via a C-terminal arm that hooks into a neighbouring molecule. This multimerization is the driving force for ATP-independent DNA unwinding by DBP during elongation. Another conserved part of DBP forms an unstructured flexible loop that is probably directly involved in contacting DNA. By making appropriate deletion mutants that do not distort the overall DBP structure, the influence of the C-terminal arm and the flexible loop on the kinetics of ssDNA binding and on DNA replication was studied. Employing surface plasmon resonance we show that both parts of the protein are required for high affinity binding. Deletion of the C-terminal arm leads to an extremely labile DBP-ssDNA complex indicating the importance of multimerization. The flexible loop is also required for optimal stability of the DBP-ssDNA complex, providing additional evidence that this region forms part of the ssDNA-binding surface of DBP. Both deletion mutants are still able to stimulate initiation of DNA replication but are defective in supporting elongation, which may be caused by the fact that both mutants have a reduced DNA unwinding activity. Surprisingly, mixtures containing both mutants do stimulate elongation. Mixing the purified mutant proteins leads to the formation of mixed filaments that have a higher affinity for ssDNA than homogeneous mutant filaments. These results provide evidence that the C-terminal arm and the flexible loop have distinct functions in unwinding during replication. We propose the following model for ATP-independent DNA unwinding by DBP. Multimerization via the C-terminal arm is required for the formation of a protein filament that saturates the displaced strand. A high affinity of a DBP monomer for ssDNA and subsequent local destabilization of the replication fork requires the flexible loop.Copyright 1998 Academic Press Limited.
|ISSN : ||0022-2836|
|Mesh Heading : ||Adenosine Triphosphate Adenoviridae Animals Base Sequence Binding Sites Biosensing Techniques DNA Primers DNA Replication DNA, Single-Stranded DNA, Viral DNA-Binding Proteins Nucleic Acid Conformation Protein Conformation Recombinant Proteins Sequence Deletion Viral Proteins metabolism genetics genetics chemistry genetics metabolism chemistry genetics chemistry genetics chemistry genetics metabolism chemistry genetics|
|Mesh Heading Relevant : ||metabolism metabolism metabolism metabolism|
Multimerization of the adenovirus DNA-binding protein is the driving force for ATP-independent DNA unwinding during strand displacement synthesis.
Journal - The EMBO journal (ENGLAND )
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.
|ISSN : ||0261-4189|
|Mesh Heading : ||Adenosine Triphosphate Adenoviridae Animals Baculoviridae Base Sequence Cell Line DNA Primers DNA, Single-Stranded DNA, Viral DNA-Binding Proteins Models, Molecular Molecular Structure Mutation Nucleic Acid Conformation Protein Conformation Sequence Deletion Spodoptera Viral Proteins metabolism genetics genetics genetics chemistry metabolism biosynthesis chemistry genetics genetics|
|Mesh Heading Relevant : ||DNA Replication metabolism chemistry metabolism chemistry metabolism|
Two regions within the DNA binding domain of nuclear factor I interact with DNA and stimulate adenovirus DNA replication independently.
Journal - Molecular and cellular biology (UNITED STATES )
The cellular transcription factor nuclear factor I (NFI) stimulates adenovirus DNA replication by up to 50-fold. The NFI DNA binding domain (NFI-BD) is sufficient for stimulation and interacts with the viral DNA polymerase, thereby recruiting the precursor terminal protein-DNA polymerase complex (pTP-pol) to the origin of replication. The mechanism of DNA binding by NFI is unknown. To examine DNA binding and stimulation of adenovirus DNA replication by NFI-BD in more detail, we generated a series of deletion mutants and show that the DNA binding domain of NFI consists of two subdomains: a highly basic N-terminal domain that binds nonspecifically to DNA and a C-terminal domain that binds specifically but with very low affinity to the NFI recognition site. Both of these subdomains stimulate DNA replication, although not to the same extent as the intact DNA binding domain. The N-terminal domain has an alpha-helical structure, as shown by circular dichroism spectroscopy. The C-terminal domain interacts with the pTP-pol complex and is able to recruit the pTP-pol complex to DNA, which leads to pTP-pol-dependent stimulation of replication. The N-terminal domain also stimulates replication in a pTP-pol-dependent manner and enhances binding of pTP-pol to DNA. Since we could not detect a direct protein-protein interaction between pTP-pol and the N-terminal domain, we suggest that this domain stimulates replication by inducing structural changes in the DNA.
|ISSN : ||0270-7306|
|Mesh Heading : ||Adenoviruses, Human Animals Base Sequence Binding Sites DNA, Viral DNA-Binding Proteins Gene Expression Regulation, Viral Molecular Sequence Data NFI Transcription Factors Nuclear Proteins Oligodeoxyribonucleotides Protein Folding RNA, Messenger Rats Recombinant Proteins Sequence Deletion Structure-Activity Relationship Virus Replication Y-Box-Binding Protein 1 biosynthesis chemistry genetics|
|Mesh Heading Relevant : ||CCAAT-Enhancer-Binding Proteins DNA Replication Transcription Factors genetics genetics|