Science Research Ideas:
Year 2002
“Metazoan origins (of replication) are still less well defined and can extend over thousands of base pairs of DNA.” (Bell & Dutta, 2002)
“The origins controlling the amplification of chorion DNA in Drosophila follicle cells and the replication of the _-globin gene cluster have been defined at the level of specific cis-acting sequences, although these sequences are relatively large compared to those defined in yeast. A number of other origins have been identified by physical analysis of replication intermediates, but less is known about specific sequences required for their function.” (Bell & Dutta, 2002)
“Although it is clear that some sites consistently act as origins of replication in the majority of eukaryotic cells, the mechanisms that select these sites and the sequences that determine their location remain elusive in many cell types.” (Bell & Dutta, 2002)
“The best-understood activity of ORC is its ability to bind DNA. Because of the availability of a known binding site, the characterization of ORC binding to DNA is most advanced for S. cerevisiae. ScORC interacts specifically with both the A and B1 elements of yeast origins of replication, spanning a region of _30 bp. Interestingly, binding to these sequences requires ATP . More precise studies of this interaction suggest ScORC interacts primarily with the A-rich strand of this region. The binding sites for other ORC complexes are less clear.” (Bell & Dutta, 2002)
“As with ScORC binding to double-stranded DNA (dsDNA) at origins, the domain of ScORC required for ssDNA binding is unknown but requires Orc1–5p.” (Bell & Dutta, 2002)
“Findings suggest that the six-subunit HsORC may be assembled on the chromatin only during the G1 and S phase of the cell cycle. This hypothesis also suggests that a more limited complex of HsORC subunits is competent for chromatin binding, although the specificity of this interaction is unclear.” (Bell & Dutta, 2002)
“Similar to DmORC association with dE2F, HsORC has been shown to interact with the viral origin binding protein, EBNA1. It is not clear, however, whether this interaction serves to localize HsORC to OriP.” (Bell & Dutta, 2002)
“The mechanism that limits the amount of ORC binding is unknown.” (Bell & Dutta, 2002)
“Mutations in the Walker A-motif of ScCdc6p result in alleles that exhibit either reduced or no function in vivo. Analysis of a mutation in the Walker B-motif shows that it either has no effect or results in a protein (Cdc6-d1p) that has reduced function when expressed in single copy but that is dominant lethal when over expressed. The mechanism of action of this dominant negative allele is unclear.” (Bell & Dutta, 2002)
“It is also not clear whether Cdc6-d1p acts dominantly by loading at the origin and preventing subsequent steps in pre-RC formation or by titrating one or more replication factors away from the origin.” (Bell & Dutta, 2002)
“Sensor I and II mutations in the SpCdc6p result in nonfunctional proteins. Thus, although these domains are critical for Cdc6p function, their exact role remains unclear.” (Bell & Dutta, 2002)
“As with Cdc6p, the function of Cdt1p in the recruitment of the MCMs to the chromatin remains unclear, although it is clear that Cdt1p is the target of the replication inhibitor geminin.” (Bell & Dutta, 2002)
“Although it is clear that the MCM proteins act at both the replication fork and origin, their biochemical function at these sites remains undetermined.” (Bell & Dutta, 2002)
“ORC, Cdc6p, and Cdt1p together are required for the stable association of the MCM proteins with the origin during G1. Although studies in a number of different organisms support the basic outline of these events, the molecular details of the recruitment of these different factors to the origin remain unclear.” (Bell & Dutta, 2002)
“The requirement for ORC function during late-origin activation has not been assessed.” (Bell & Dutta, 2002)
“CDK modification also controls the function of the MCM proteins, although the mechanism of this control is unclear. Invitro, CDK can phosphorylate at least Mcm2p and Mcm4p, and the same subunits are phosphorylated in vivo. For the most part the exact sites of phosphorylation have not been mapped; however, the mutation of six consensus CDK sites on mouse Mcm4p prevented phosphorylation by cyclin A/Cdk2 or cyclin B/Cdk1, and at least one of these sites is phosphorylated in a cell-cycle-dependent manner in vivo ” (Bell & Dutta, 2002)
“The molecular consequences of phosphorylation of yeast ORC remain unclear.” (Bell & Dutta, 2002)
“Unlike the situation for Cdc6p and the MCM proteins, the aspect of ORC function inhibited by CDK phosphorylation is unclear.” (Bell & Dutta, 2002)
“All the events of DNA replication must also contend with chromatin-assembled templates, yet the effects of chromatin on replication are relatively unclear at a molecular level.” (Bell & Dutta, 2002)
“If pre-RC formation is not sufficient for passage through the ODP (Origin Decision Point), the event responsible for activating only a specific subset of pre-RCs formed in telophase remains unknown.” (Bell & Dutta, 2002)
“What remains unclear, however, is when in the cell cycle Mcm10p is associated with the origin and when it is associated with MCM proteins.” (Bell & Dutta, 2002)
“This suggests that Mcm10p functions at the transition from the pre-RC to the elongating state. Although XlMcm10p is not required for DNA synthesis on M13 ssDNA, it remains an open question as to whether it functions at the intact replication fork.” (Bell & Dutta, 2002)
“Cdc45p is required for the assembly of many components of the DNA synthetic machinery at the replication fork, including RPA, PCNA, and DNA polymerases α and ε. Although the exact order of assembly of these remaining components is unclear.” (Bell & Dutta, 2002)
“In vivo phosphorylation studies in S. cerevisiae indicate that five of the six MCM proteins are phosphorylated, but the cell cycle regulation of this phosphorylation is not well characterized.” (Bell & Dutta, 2002)
“The ability of this mutant Cdc6p to recruit MCMs and cyclin E-Cdk2 to chromatin in these assays was not tested, bhowever, leaving open the possibility that the defect induced by this mutation is involved in other aspects of Cdc6p function besides cyclin E-Cdk2 recruitment (e.g., XlMCM loading).” (Bell & Dutta, 2002)
“Human Orc1p physically associates with cyclin E-Cdk2 and cyclin A-Cdk1 in vitro (A. Dutta, unpublished data). The region of ORC required for these interactions in unknown; however, there is a Cy-motif in the human and Xenopus Orc1 proteins that could be responsible for the interaction. Although intriguing, these interactions have yet to be tested for relevance in any organism.” (Bell & Dutta, 2002)
“There is an absolute requirement for CDK activity for DNA replication, yet the proteins that must be phosphorylated to promote replication remain unknown.” (Bell & Dutta, 2002)
“It has not been demonstrated that phosphorylation by CDK activates the kinase.” (Bell & Dutta, 2002)
“The time at which a eukaryotic origin initiates replication within S phase is a characteristic of each origin. Although the mechanisms that control this regulation are largely a mystery, there are a number of interesting clues.” (Bell & Dutta, 2002)
“Although how chromatin structure and local transcription affect the assembly of proteins at the origin is unclear, the differences between replication factor assembly at these sites are well documented.” (Bell & Dutta, 2002)
“Rad53p appears to promote the expression of Dbf4p mRNA and protein through unknown mechanisms.” (Bell & Dutta, 2002)
“How origins are selected in species other than S. cerevisiae remains largely undetermined.” (Bell & Dutta, 2002)
“Although currently unknown, the determinants of the origin decision point (ODP) are likely to be key contributors to the origin selection process.” (Bell & Dutta, 2002)
“Major progress has been made recently in identifying the proteins that are recruited to the origin and the order in which they are recruited; however, our knowledge of their biochemical activities once they arrive at the origin is still very limited.” (Bell & Dutta, 2002)
“Although Cdc45p and/or Mcm10p are required for the opening of the DNA duplex and recruitment of the polymerases,
it remains unclear what the biochemical roles of these factors are in these events. Do they stimulate other proteins to unwind the DNA or do they directly participate in this event? Are they in direct contact with all of the polymerases and/or the ssDNA? Equally unclear is the mechanism by which these events are activated by the CDKs and DDKs. Again, the requirement of these kinases to trigger the key events of the transition to replication is undisputed, yet the substrates that are modified and the consequences of these modifications remain largely unknown.” (Bell & Dutta, 2002)
References:
Bell, S. P., & Dutta, A. (2002). DNA REPLICATION IN EUKARYOTIC CELLS. Annu. Rev. Biochem. , 333-374.
“The origins controlling the amplification of chorion DNA in Drosophila follicle cells and the replication of the _-globin gene cluster have been defined at the level of specific cis-acting sequences, although these sequences are relatively large compared to those defined in yeast. A number of other origins have been identified by physical analysis of replication intermediates, but less is known about specific sequences required for their function.” (Bell & Dutta, 2002)
“Although it is clear that some sites consistently act as origins of replication in the majority of eukaryotic cells, the mechanisms that select these sites and the sequences that determine their location remain elusive in many cell types.” (Bell & Dutta, 2002)
“The best-understood activity of ORC is its ability to bind DNA. Because of the availability of a known binding site, the characterization of ORC binding to DNA is most advanced for S. cerevisiae. ScORC interacts specifically with both the A and B1 elements of yeast origins of replication, spanning a region of _30 bp. Interestingly, binding to these sequences requires ATP . More precise studies of this interaction suggest ScORC interacts primarily with the A-rich strand of this region. The binding sites for other ORC complexes are less clear.” (Bell & Dutta, 2002)
“As with ScORC binding to double-stranded DNA (dsDNA) at origins, the domain of ScORC required for ssDNA binding is unknown but requires Orc1–5p.” (Bell & Dutta, 2002)
“Findings suggest that the six-subunit HsORC may be assembled on the chromatin only during the G1 and S phase of the cell cycle. This hypothesis also suggests that a more limited complex of HsORC subunits is competent for chromatin binding, although the specificity of this interaction is unclear.” (Bell & Dutta, 2002)
“Similar to DmORC association with dE2F, HsORC has been shown to interact with the viral origin binding protein, EBNA1. It is not clear, however, whether this interaction serves to localize HsORC to OriP.” (Bell & Dutta, 2002)
“The mechanism that limits the amount of ORC binding is unknown.” (Bell & Dutta, 2002)
“Mutations in the Walker A-motif of ScCdc6p result in alleles that exhibit either reduced or no function in vivo. Analysis of a mutation in the Walker B-motif shows that it either has no effect or results in a protein (Cdc6-d1p) that has reduced function when expressed in single copy but that is dominant lethal when over expressed. The mechanism of action of this dominant negative allele is unclear.” (Bell & Dutta, 2002)
“It is also not clear whether Cdc6-d1p acts dominantly by loading at the origin and preventing subsequent steps in pre-RC formation or by titrating one or more replication factors away from the origin.” (Bell & Dutta, 2002)
“Sensor I and II mutations in the SpCdc6p result in nonfunctional proteins. Thus, although these domains are critical for Cdc6p function, their exact role remains unclear.” (Bell & Dutta, 2002)
“As with Cdc6p, the function of Cdt1p in the recruitment of the MCMs to the chromatin remains unclear, although it is clear that Cdt1p is the target of the replication inhibitor geminin.” (Bell & Dutta, 2002)
“Although it is clear that the MCM proteins act at both the replication fork and origin, their biochemical function at these sites remains undetermined.” (Bell & Dutta, 2002)
“ORC, Cdc6p, and Cdt1p together are required for the stable association of the MCM proteins with the origin during G1. Although studies in a number of different organisms support the basic outline of these events, the molecular details of the recruitment of these different factors to the origin remain unclear.” (Bell & Dutta, 2002)
“The requirement for ORC function during late-origin activation has not been assessed.” (Bell & Dutta, 2002)
“CDK modification also controls the function of the MCM proteins, although the mechanism of this control is unclear. Invitro, CDK can phosphorylate at least Mcm2p and Mcm4p, and the same subunits are phosphorylated in vivo. For the most part the exact sites of phosphorylation have not been mapped; however, the mutation of six consensus CDK sites on mouse Mcm4p prevented phosphorylation by cyclin A/Cdk2 or cyclin B/Cdk1, and at least one of these sites is phosphorylated in a cell-cycle-dependent manner in vivo ” (Bell & Dutta, 2002)
“The molecular consequences of phosphorylation of yeast ORC remain unclear.” (Bell & Dutta, 2002)
“Unlike the situation for Cdc6p and the MCM proteins, the aspect of ORC function inhibited by CDK phosphorylation is unclear.” (Bell & Dutta, 2002)
“All the events of DNA replication must also contend with chromatin-assembled templates, yet the effects of chromatin on replication are relatively unclear at a molecular level.” (Bell & Dutta, 2002)
“If pre-RC formation is not sufficient for passage through the ODP (Origin Decision Point), the event responsible for activating only a specific subset of pre-RCs formed in telophase remains unknown.” (Bell & Dutta, 2002)
“What remains unclear, however, is when in the cell cycle Mcm10p is associated with the origin and when it is associated with MCM proteins.” (Bell & Dutta, 2002)
“This suggests that Mcm10p functions at the transition from the pre-RC to the elongating state. Although XlMcm10p is not required for DNA synthesis on M13 ssDNA, it remains an open question as to whether it functions at the intact replication fork.” (Bell & Dutta, 2002)
“Cdc45p is required for the assembly of many components of the DNA synthetic machinery at the replication fork, including RPA, PCNA, and DNA polymerases α and ε. Although the exact order of assembly of these remaining components is unclear.” (Bell & Dutta, 2002)
“In vivo phosphorylation studies in S. cerevisiae indicate that five of the six MCM proteins are phosphorylated, but the cell cycle regulation of this phosphorylation is not well characterized.” (Bell & Dutta, 2002)
“The ability of this mutant Cdc6p to recruit MCMs and cyclin E-Cdk2 to chromatin in these assays was not tested, bhowever, leaving open the possibility that the defect induced by this mutation is involved in other aspects of Cdc6p function besides cyclin E-Cdk2 recruitment (e.g., XlMCM loading).” (Bell & Dutta, 2002)
“Human Orc1p physically associates with cyclin E-Cdk2 and cyclin A-Cdk1 in vitro (A. Dutta, unpublished data). The region of ORC required for these interactions in unknown; however, there is a Cy-motif in the human and Xenopus Orc1 proteins that could be responsible for the interaction. Although intriguing, these interactions have yet to be tested for relevance in any organism.” (Bell & Dutta, 2002)
“There is an absolute requirement for CDK activity for DNA replication, yet the proteins that must be phosphorylated to promote replication remain unknown.” (Bell & Dutta, 2002)
“It has not been demonstrated that phosphorylation by CDK activates the kinase.” (Bell & Dutta, 2002)
“The time at which a eukaryotic origin initiates replication within S phase is a characteristic of each origin. Although the mechanisms that control this regulation are largely a mystery, there are a number of interesting clues.” (Bell & Dutta, 2002)
“Although how chromatin structure and local transcription affect the assembly of proteins at the origin is unclear, the differences between replication factor assembly at these sites are well documented.” (Bell & Dutta, 2002)
“Rad53p appears to promote the expression of Dbf4p mRNA and protein through unknown mechanisms.” (Bell & Dutta, 2002)
“How origins are selected in species other than S. cerevisiae remains largely undetermined.” (Bell & Dutta, 2002)
“Although currently unknown, the determinants of the origin decision point (ODP) are likely to be key contributors to the origin selection process.” (Bell & Dutta, 2002)
“Major progress has been made recently in identifying the proteins that are recruited to the origin and the order in which they are recruited; however, our knowledge of their biochemical activities once they arrive at the origin is still very limited.” (Bell & Dutta, 2002)
“Although Cdc45p and/or Mcm10p are required for the opening of the DNA duplex and recruitment of the polymerases,
it remains unclear what the biochemical roles of these factors are in these events. Do they stimulate other proteins to unwind the DNA or do they directly participate in this event? Are they in direct contact with all of the polymerases and/or the ssDNA? Equally unclear is the mechanism by which these events are activated by the CDKs and DDKs. Again, the requirement of these kinases to trigger the key events of the transition to replication is undisputed, yet the substrates that are modified and the consequences of these modifications remain largely unknown.” (Bell & Dutta, 2002)
References:
Bell, S. P., & Dutta, A. (2002). DNA REPLICATION IN EUKARYOTIC CELLS. Annu. Rev. Biochem. , 333-374.
