Science Research Ideas
Biochemistry
2010:
One of the most striking features of DNA replication in higher eukaryotes is its plasticity. A best-known example of plasticity is the very rapid replication of the entire genome of the fertilized eggs of amphibians (within a matter of 20–30 min), compared with the 8–10 h required for the genome replication in somatic cells. This robust process is achieved by very frequent initiation through utilization of virtually all the potential origins in contrast to the spatially and temporally regulated origin firing that occurs in differentiated cells. In spite of this, how this rather dramatic transition of the replication mode takes place is still under debate. (Masai, Matsumoto, You, Sugata, & Oda, 2010)
UV-footprinting analyses showed that the pre-RC to post-RC conversion is observed at suppressed late origins after passage of the replication fork from neighboring origins. By analyses of single DNA molecules, it was demonstrated that this so-called origin interference does occur in human cells as well, maintaining a relatively constant interorigin distance. The precise mechanisms of origin interference are not known, although passive replication may well play a significant role in the phenomenon. (Masai, Matsumoto, You, Sugata, & Oda, 2010)
It has been generally assumed that stalling of the replication fork by hydroxyurea or aphidicolin, the condition used to slow the fork rate in the above experiments, induces checkpoint reactions and inhibits further origin firing. It is unclear how the same treatment can induce opposite outcome. (Masai, Matsumoto, You, Sugata, & Oda, 2010)
In fission yeast, the definition of early- and late-firing origins is less clear. There is certainly a set of efficient origins that fire in the presence of hydroxyurea, and other origins are inefficient and normally do not fire under the same conditions. It is not known whether these inefficient origins actually fire during the normal course of S phase or are passively replicated, although Cdc45 loading may take place in late S phase at some of these inefficient origins. (Masai, Matsumoto, You, Sugata, & Oda, 2010)
The mechanisms that regulate replication timing in metazoans remain elusive. (Masai, Matsumoto, You, Sugata, & Oda, 2010)
“Incubation of nuclei from differentiated cells at various cell cycle stages in Xenopus egg extracts indicated that nuclei fromMor early G1 phases replicate in an “embryonic” manner, whereas those from after the TDP (timing decision point) within the G1 phase replicate with a unique timing regulation (in a differentiated manner). However, the nature of the transition that occurs at theTDPis unknown. It was proposed that nuclear structures may play a role in determining the timing of DNA replication.” (Masai, Matsumoto, You, Sugata, & Oda, 2010)
“The precise roles of c-Myc (a frequently deregulated proto-oncogene) in regulation of the pre-RC remain to be investigated. Phosphorylation of the N-terminal segment of MCM2 by the Cdc7 kinase was reported to be important for pre-RC formation during the quiescence to S phase transition , although it is not known whether this regulation operates during the G1 phase of proliferating cells.” (Masai, Matsumoto, You, Sugata, & Oda, 2010)
“Although the human ortholog of Sld3 has not been identified yet, the interaction of replication proteins dependent on CDK-mediated phosphorylation might be well conserved. Recently, a vertebrate factor with functions potentially related to Sld3 were reported.” (Masai, Matsumoto, You, Sugata, & Oda, 2010)
“It is yet to be determined whether the MCM2∼7 complexes from other species exhibit DNA helicase activity under similar conditions.” (Masai, Matsumoto, You, Sugata, & Oda, 2010)
Although G1 signaling via Rb and CDKs influences DNA replication, several recent studies also suggest that appropriate and timely activation of G1 CDKs and G1/S-phase progression is critically dependent on replication licensing. It was shown that impaired pre-RC assembly delays G1 phase progression via multiple mechanisms, including reduced transcriptional induction of Cyclin D1 (the activating binding partner of Cdk4/6), induction of CDK inhibitors p21 and p27, and p53-dependent loss of Thr160 phosphorylation of Cdk2 required for its activation. Further studies are necessary to determine the precise mechanisms of these novel licensing checkpoints. (Masai, Matsumoto, You, Sugata, & Oda, 2010)
Although PriA (A protein in Bacteria) is widely conserved in eubacterial species, structural and functional homologs of PriA in other kingdoms have not been identified yet, and it is not clear whether similar mechanisms operate in eukaryotes. (Masai, Matsumoto, You, Sugata, & Oda, 2010)
The putative mechanisms by which ATR/Chk1 (ATR is ataxia-telangiectasia-mutated and Rad3- Relatedand Chk1 is a protein in checkpoint) signaling may negatively regulate Cdc7-Dbf4 in Xenopus(a genus of frogs) are not known. (Masai, Matsumoto, You, Sugata, & Oda, 2010)
It is not yet clear whether Cdc7 is a target of the intra-S checkpoint in all experimental systems. (Masai, Matsumoto, You, Sugata, & Oda, 2010)
It is still not clear at this stage (G1 phase) exactly what kind of mark is left on the chromatin or what kind of higher chromatin structures are formed to differentiate the initiation sites and timing of replication during the S phase. (Masai, Matsumoto, You, Sugata, & Oda, 2010)
Localization of the pre-RC was examined genome wide in yeasts and Drosophila, and enrichment in the promoter region was observed, suggesting that the promoter regions provide a favorable chromatin environment for pre-RC assembly. It remains to be seen whether pre-RCs are assembled in a similar manner in mammals. (Masai, Matsumoto, You, Sugata, & Oda, 2010)
How is the site-specific initiation achieved? In other words, how are origins differentially selected from the list of pre-RCs? Alternatively, is this selection truly stochastic? (Masai, Matsumoto, You, Sugata, & Oda, 2010)
What are the exact mechanisms that determine the timing of origin firing? Are there any novel factors involved in defining the timing pattern of potential origins or setting the replication timing domains? (Masai, Matsumoto, You, Sugata, & Oda, 2010)
What are the mechanisms that coordinate fork rate and new origin firing during the S phase, and how are the stalled fork signals transmitted to activate dormant origins? (Masai, Matsumoto, You, Sugata, & Oda, 2010)
What is the nature of the active replicative DNA helicase at the eukaryotic replication forks and how does it operate? (Masai, Matsumoto, You, Sugata, & Oda, 2010)
What is the molecular architecture of the eukaryotic replication fork? Could assembly of a replication fork and initiation of DNA replication be reconstituted with purified proteins? (Masai, Matsumoto, You, Sugata, & Oda, 2010)
How is the replication fork reorganized upon encounter with fork blocks, and what are the exact mechanisms for inhibition of new firing, slowing the replication fork, and the eventual reinitiation of DNA replication? (Masai, Matsumoto, You, Sugata, & Oda, 2010)
How do aberrations in replication and checkpoint factors lead to various diseases, and what is the molecular basis of cancer cell–specific cell death induced by inhibition of replication factors? (Masai, Matsumoto, You, Sugata, & Oda, 2010)
How are the replication cycle and checkpoint regulation coordinated with the circadian cycle or metabolic cycle? Are there potential “extra-DNA replication” functions of replication factors? (Masai, Matsumoto, You, Sugata, & Oda, 2010)
One of the most striking features of DNA replication in higher eukaryotes is its plasticity. A best-known example of plasticity is the very rapid replication of the entire genome of the fertilized eggs of amphibians (within a matter of 20–30 min), compared with the 8–10 h required for the genome replication in somatic cells. This robust process is achieved by very frequent initiation through utilization of virtually all the potential origins in contrast to the spatially and temporally regulated origin firing that occurs in differentiated cells. In spite of this, how this rather dramatic transition of the replication mode takes place is still under debate. (Masai, Matsumoto, You, Sugata, & Oda, 2010)
UV-footprinting analyses showed that the pre-RC to post-RC conversion is observed at suppressed late origins after passage of the replication fork from neighboring origins. By analyses of single DNA molecules, it was demonstrated that this so-called origin interference does occur in human cells as well, maintaining a relatively constant interorigin distance. The precise mechanisms of origin interference are not known, although passive replication may well play a significant role in the phenomenon. (Masai, Matsumoto, You, Sugata, & Oda, 2010)
It has been generally assumed that stalling of the replication fork by hydroxyurea or aphidicolin, the condition used to slow the fork rate in the above experiments, induces checkpoint reactions and inhibits further origin firing. It is unclear how the same treatment can induce opposite outcome. (Masai, Matsumoto, You, Sugata, & Oda, 2010)
In fission yeast, the definition of early- and late-firing origins is less clear. There is certainly a set of efficient origins that fire in the presence of hydroxyurea, and other origins are inefficient and normally do not fire under the same conditions. It is not known whether these inefficient origins actually fire during the normal course of S phase or are passively replicated, although Cdc45 loading may take place in late S phase at some of these inefficient origins. (Masai, Matsumoto, You, Sugata, & Oda, 2010)
The mechanisms that regulate replication timing in metazoans remain elusive. (Masai, Matsumoto, You, Sugata, & Oda, 2010)
“Incubation of nuclei from differentiated cells at various cell cycle stages in Xenopus egg extracts indicated that nuclei fromMor early G1 phases replicate in an “embryonic” manner, whereas those from after the TDP (timing decision point) within the G1 phase replicate with a unique timing regulation (in a differentiated manner). However, the nature of the transition that occurs at theTDPis unknown. It was proposed that nuclear structures may play a role in determining the timing of DNA replication.” (Masai, Matsumoto, You, Sugata, & Oda, 2010)
“The precise roles of c-Myc (a frequently deregulated proto-oncogene) in regulation of the pre-RC remain to be investigated. Phosphorylation of the N-terminal segment of MCM2 by the Cdc7 kinase was reported to be important for pre-RC formation during the quiescence to S phase transition , although it is not known whether this regulation operates during the G1 phase of proliferating cells.” (Masai, Matsumoto, You, Sugata, & Oda, 2010)
“Although the human ortholog of Sld3 has not been identified yet, the interaction of replication proteins dependent on CDK-mediated phosphorylation might be well conserved. Recently, a vertebrate factor with functions potentially related to Sld3 were reported.” (Masai, Matsumoto, You, Sugata, & Oda, 2010)
“It is yet to be determined whether the MCM2∼7 complexes from other species exhibit DNA helicase activity under similar conditions.” (Masai, Matsumoto, You, Sugata, & Oda, 2010)
Although G1 signaling via Rb and CDKs influences DNA replication, several recent studies also suggest that appropriate and timely activation of G1 CDKs and G1/S-phase progression is critically dependent on replication licensing. It was shown that impaired pre-RC assembly delays G1 phase progression via multiple mechanisms, including reduced transcriptional induction of Cyclin D1 (the activating binding partner of Cdk4/6), induction of CDK inhibitors p21 and p27, and p53-dependent loss of Thr160 phosphorylation of Cdk2 required for its activation. Further studies are necessary to determine the precise mechanisms of these novel licensing checkpoints. (Masai, Matsumoto, You, Sugata, & Oda, 2010)
Although PriA (A protein in Bacteria) is widely conserved in eubacterial species, structural and functional homologs of PriA in other kingdoms have not been identified yet, and it is not clear whether similar mechanisms operate in eukaryotes. (Masai, Matsumoto, You, Sugata, & Oda, 2010)
The putative mechanisms by which ATR/Chk1 (ATR is ataxia-telangiectasia-mutated and Rad3- Relatedand Chk1 is a protein in checkpoint) signaling may negatively regulate Cdc7-Dbf4 in Xenopus(a genus of frogs) are not known. (Masai, Matsumoto, You, Sugata, & Oda, 2010)
It is not yet clear whether Cdc7 is a target of the intra-S checkpoint in all experimental systems. (Masai, Matsumoto, You, Sugata, & Oda, 2010)
It is still not clear at this stage (G1 phase) exactly what kind of mark is left on the chromatin or what kind of higher chromatin structures are formed to differentiate the initiation sites and timing of replication during the S phase. (Masai, Matsumoto, You, Sugata, & Oda, 2010)
Localization of the pre-RC was examined genome wide in yeasts and Drosophila, and enrichment in the promoter region was observed, suggesting that the promoter regions provide a favorable chromatin environment for pre-RC assembly. It remains to be seen whether pre-RCs are assembled in a similar manner in mammals. (Masai, Matsumoto, You, Sugata, & Oda, 2010)
How is the site-specific initiation achieved? In other words, how are origins differentially selected from the list of pre-RCs? Alternatively, is this selection truly stochastic? (Masai, Matsumoto, You, Sugata, & Oda, 2010)
What are the exact mechanisms that determine the timing of origin firing? Are there any novel factors involved in defining the timing pattern of potential origins or setting the replication timing domains? (Masai, Matsumoto, You, Sugata, & Oda, 2010)
What are the mechanisms that coordinate fork rate and new origin firing during the S phase, and how are the stalled fork signals transmitted to activate dormant origins? (Masai, Matsumoto, You, Sugata, & Oda, 2010)
What is the nature of the active replicative DNA helicase at the eukaryotic replication forks and how does it operate? (Masai, Matsumoto, You, Sugata, & Oda, 2010)
What is the molecular architecture of the eukaryotic replication fork? Could assembly of a replication fork and initiation of DNA replication be reconstituted with purified proteins? (Masai, Matsumoto, You, Sugata, & Oda, 2010)
How is the replication fork reorganized upon encounter with fork blocks, and what are the exact mechanisms for inhibition of new firing, slowing the replication fork, and the eventual reinitiation of DNA replication? (Masai, Matsumoto, You, Sugata, & Oda, 2010)
How do aberrations in replication and checkpoint factors lead to various diseases, and what is the molecular basis of cancer cell–specific cell death induced by inhibition of replication factors? (Masai, Matsumoto, You, Sugata, & Oda, 2010)
How are the replication cycle and checkpoint regulation coordinated with the circadian cycle or metabolic cycle? Are there potential “extra-DNA replication” functions of replication factors? (Masai, Matsumoto, You, Sugata, & Oda, 2010)
