scholarly journals Differences in the Single-stranded DNA Binding Activities of MCM2-7 and MCM467

2007 ◽  
Vol 282 (46) ◽  
pp. 33795-33804 ◽  
Author(s):  
Matthew L. Bochman ◽  
Anthony Schwacha
Keyword(s):  
Dna Binding ◽  
Active Site ◽  
Biochemical Properties ◽  
Dna Helicase ◽  
Binding Activity ◽  
Helicase Activity ◽  
Single Stranded Dna ◽  
Ssdna Binding ◽  
Better Than

The MCM2-7 complex, a hexamer containing six distinct and essential subunits, is postulated to be the eukaryotic replicative DNA helicase. Although all six subunits function at the replication fork, only a specific subcomplex consisting of the MCM4, 6, and 7 subunits (MCM467) and not the MCM2-7 complex exhibits DNA helicase activity in vitro. To understand why MCM2-7 lacks helicase activity and to address the possible function of the MCM2, 3, and 5 subunits, we have compared the biochemical properties of the Saccharomyces cerevisiae MCM2-7 and MCM467 complexes. We demonstrate that both complexes are toroidal and possess a similar ATP-dependent single-stranded DNA (ssDNA) binding activity, indicating that the lack of helicase activity by MCM2-7 is not due to ineffective ssDNA binding. We identify two important differences between them. MCM467 binds dsDNA better than MCM2-7. In addition, we find that the rate of MCM2-7/ssDNA association is slow compared with MCM467; the association rate can be dramatically increased either by preincubation with ATP or by inclusion of mutations that ablate the MCM2/5 active site. We propose that the DNA binding differences between MCM2-7 and MCM467 correspond to a conformational change at the MCM2/5 active site with putative regulatory significance.

scholarly journals Biochemical Analysis of the Intrinsic Mcm4-Mcm6-Mcm7 DNA Helicase Activity

1999 ◽  
Vol 19 (12) ◽  
pp. 8003-8015 ◽  
Author(s):  
Zhiying You ◽  
Yuki Komamura ◽  
Yukio Ishimi
Keyword(s):  
Dna Replication ◽  
Dna Binding ◽  
Dna Helicase ◽  
Binding Activity ◽  
Helicase Activity ◽  
Single Stranded Dna ◽  
Dna Binding Activity ◽  
Eukaryotic Dna ◽  
Mcm Proteins ◽  
Biochemical Analyses

ABSTRACT Mcm proteins play an essential role in eukaryotic DNA replication, but their biochemical functions are poorly understood. Recently, we reported that a DNA helicase activity is associated with an Mcm4-Mcm6-Mcm7 (Mcm4,6,7) complex, suggesting that this complex is involved in the initiation of DNA replication as a DNA-unwinding enzyme. In this study, we have expressed and isolated the mouse Mcm2,4,6,7 proteins from insect cells and characterized various mutant Mcm4,6,7 complexes in which the conserved ATPase motifs of the Mcm4 and Mcm6 proteins were mutated. The activities associated with such preparations demonstrated that the DNA helicase activity is intrinsically associated with the Mcm4,6,7 complex. Biochemical analyses of these mutant Mcm4,6,7 complexes indicated that the ATP binding activity of the Mcm6 protein in the complex is critical for DNA helicase activity and that the Mcm4 protein may play a role in the single-stranded DNA binding activity of the complex. The results also indicated that the two activities of DNA helicase and single-stranded DNA binding can be separated.

scholarly journals Parvovirus Initiator Protein NS1 and RPA Coordinate Replication Fork Progression in a Reconstituted DNA Replication System

2002 ◽  
Vol 76 (13) ◽  
pp. 6518-6531 ◽  
Author(s):  
Jesper Christensen ◽  
Peter Tattersall
Keyword(s):  
Dna Binding ◽  
Replication Fork ◽  
Binding Protein ◽  
Dna Binding Protein ◽  
Small Subunit ◽  
Dna Helicase ◽  
Enzyme Linked Immunosorbent Assay ◽  
Helicase Activity ◽  
Single Stranded Dna ◽  
Initiator Protein

ABSTRACT We show here that the DNA helicase activity of the parvoviral initiator protein NS1 is highly directional, binding to the single strand at a recessed 5′ end and displacing the other strand while progressing in a 3′-to-5′ direction on the bound strand. NS1 and a cellular site-specific DNA binding factor, PIF, also known as glucocorticoid modulating element binding protein, bind to the left-end minimal replication origin of minute virus of mice, forming a ternary complex. In this complex, NS1 is activated to nick one DNA strand, becoming covalently attached to the 5′ end of the nick in the process and providing a 3′ OH for priming DNA synthesis. In this situation, the helicase activity of NS1 did not displace the nicked strand, but the origin duplex was distorted by the NS1-PIF complex, as assayed by its sensitivity to KMnO4 oxidation, and a stretch of about 14 nucleotides on both strands of the nicked origin underwent limited unwinding. Addition of Escherichia coli single-stranded DNA binding protein (SSB) did not lead to further unwinding. However, addition of recombinant human single-stranded DNA binding protein (RPA) to the initiation reaction catalyzed extensive unwinding of the nicked origin, suggesting that RPA may be required to form a functional replication fork. Accordingly, the unwinding mediated by NS1 and RPA promoted processive leading-strand synthesis catalyzed by recombinant human DNA polymerase δ, PCNA, and RFC, using the minimal left-end origin cloned in a plasmid as a template. The requirement for RPA, rather than SSB, in the unwinding reaction indicated that specific NS1-RPA protein interactions were formed. NS1 was tested by enzyme-linked immunosorbent assay for binding to two- or three-subunit RPA complexes expressed from recombinant baculoviruses. NS1 efficiently bound each of the baculovirus-expressed complexes, indicating that the small subunit of RPA is not involved in specific NS1 binding. No NS1 interactions were observed with E. coli SSB or other proteins included as controls.

Purification of RIP60 and RIP100, mammalian proteins with origin-specific DNA-binding and ATP-dependent DNA helicase activities

1990 ◽  
Vol 10 (12) ◽  
pp. 6225-6235
Author(s):  
L Dailey ◽  
M S Caddle ◽  
N Heintz ◽  
N H Heintz
Keyword(s):  
Dna Binding ◽  
Dna Sequences ◽  
Mammalian Cells ◽  
Dna Helicase ◽  
Binding Activity ◽  
Helicase Activity ◽  
Chromosomal Dna ◽  
Bent Dna ◽  
Autonomously Replicating Sequence ◽  
Dna Binding Activity

Replication of the Chinese hamster dihydrofolate reductase gene (dhfr) initiates near a fragment of stably bent DNA that binds multiple cellular factors. Investigation of protein interactions with the dhfr bent DNA sequences revealed a novel nuclear protein that also binds to domain B of the yeast origin of replication, the autonomously replicating sequence ARS1. The origin-specific DNA-binding activity was purified 9,000-fold from HeLa cell nuclear extract in five chromatographic steps. Protein-DNA cross-linking experiments showed that a 60-kDa polypeptide, which we call RIP60, contained the origin-specific DNA-binding activity. Oligonucleotide displacement assays showed that highly purified fractions of RIP60 also contained an ATP-dependent DNA helicase activity. Covalent radiolabeling with ATP indicated that the DNA helicase activity resided in a 100-kDa polypeptide, RIP100. The cofractionation of an ATP-dependent DNA helicase with an origin-specific DNA-binding activity suggests that RIP60 and RIP100 may be involved in initiation of chromosomal DNA synthesis in mammalian cells.

scholarly journals The WYL domain of the PIF1 helicase from the thermophilic bacteriumThermotoga elfiiis an accessory single-stranded DNA binding module

2017 ◽  
Author(s):  
Nicholas M. Andis ◽  
Christopher W. Sausen ◽  
Ashna Alladin ◽  
Matthew L. Bochman
Keyword(s):  
Unknown Function ◽  
Genome Stability ◽  
Dna Helicase ◽  
Thermophilic Bacterium ◽  
Helicase Domain ◽  
Single Stranded Dna ◽  
Ssdna Binding ◽  
Pif1 Helicase

ABSTRACTPIF1 family helicases are conserved from bacteria to man. With the exception of the well-studied yeast PIF1 helicases (e.g., ScPif1 and ScRrm3), however, very little is known about how these enzymes help maintain genome stability. Indeed, we lack a basic understanding of the protein domains found N- and C-terminal to the characteristic central PIF1 helicase domain in these proteins. Here, using chimeric constructs, we show that the ScPif1 and ScRrm3 helicase domains are interchangeable and that the N-terminus of ScRrm3 is important for its functionin vivo. This suggests that PIF1 family helicases evolved functional modules fused to a generic motor domain. To investigate this hypothesis, we characterized the biochemical activities of the PIF1 helicase from the thermophilic bacteriumThermotoga elfii(TePif1), which contains a C-terminal WYL domain of unknown function. Like helicases from other thermophiles, recombinant TePif1 was easily prepared, thermostablein vitro, and displayed activities similar to its eukaryotic homologs. We also found that the WYL domain was necessary for high-affinity single-stranded DNA (ssDNA) binding and affected both ATPase and helicase activities. Deleting the WYL domain from TePif1 or mutating conserved residues in the predicted ssDNA binding site uncoupled ATPase activity and DNA unwinding, leading to higher rates of ATP hydrolysis but less efficient DNA helicase activity. Our findings suggest that the domains of unknown function found in eukaryotic PIF1 helicases may also confer functional specificity and additional activities to these enzymes, which should be investigated in future work.

scholarly journals Purification of RIP60 and RIP100, mammalian proteins with origin-specific DNA-binding and ATP-dependent DNA helicase activities.

1990 ◽  
Vol 10 (12) ◽  
pp. 6225-6235 ◽  
Author(s):  
L Dailey ◽  
M S Caddle ◽  
N Heintz ◽  
N H Heintz
Keyword(s):  
Dna Binding ◽  
Dna Sequences ◽  
Mammalian Cells ◽  
Dna Helicase ◽  
Binding Activity ◽  
Helicase Activity ◽  
Chromosomal Dna ◽  
Bent Dna ◽  
Autonomously Replicating Sequence ◽  
Dna Binding Activity

Replication of the Chinese hamster dihydrofolate reductase gene (dhfr) initiates near a fragment of stably bent DNA that binds multiple cellular factors. Investigation of protein interactions with the dhfr bent DNA sequences revealed a novel nuclear protein that also binds to domain B of the yeast origin of replication, the autonomously replicating sequence ARS1. The origin-specific DNA-binding activity was purified 9,000-fold from HeLa cell nuclear extract in five chromatographic steps. Protein-DNA cross-linking experiments showed that a 60-kDa polypeptide, which we call RIP60, contained the origin-specific DNA-binding activity. Oligonucleotide displacement assays showed that highly purified fractions of RIP60 also contained an ATP-dependent DNA helicase activity. Covalent radiolabeling with ATP indicated that the DNA helicase activity resided in a 100-kDa polypeptide, RIP100. The cofractionation of an ATP-dependent DNA helicase with an origin-specific DNA-binding activity suggests that RIP60 and RIP100 may be involved in initiation of chromosomal DNA synthesis in mammalian cells.

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