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Anatoliy Li

Publications and source records attributed to Anatoliy Li.

8 recordsLinked to original sources
PubMed2006

Deregulated replication licensing causes DNA fragmentation consistent with head-to-tail fork collision.

Correct regulation of the replication licensing system ensures that no DNA is rereplicated in a single cell cycle. When the licensing protein Cdt1 is overexpressed in G2 phase of the cell cycle, replication origins are relicensed and the DNA is rereplicated. At the same time, checkpoint pathways are activated that block further cell cycle progression. We have studied the consequence of deregulating the licensing system by adding recombinant Cdt1 to Xenopus egg extracts. We show that Cdt1 induces checkpoint activation and the appearance of small fragments of double-stranded DNA. DNA fragmentation and strong checkpoint activation are dependent on uncontrolled rereplication and do not occur after a single coordinated round of rereplication. The DNA fragments are composed exclusively of rereplicated DNA. The unusual characteristics of these fragments suggest that they result from head-to-tail collision (rear ending) of replication forks chasing one another along the same DNA template.

Animals↗
PubMed2005

Functional domains of the Xenopus replication licensing factor Cdt1.

During late mitosis and early G1, replication origins are licensed for subsequent replication by loading heterohexamers of the mini-chromosome maintenance proteins (Mcm2-7). To prevent re-replication of DNA, the licensing system is down-regulated at other cell cycle stages. A small protein called geminin plays an important role in this down-regulation by binding and inhibiting the Cdt1 component of the licensing system. We examine here the organization of Xenopus Cdt1, delimiting regions of Cdt1 required for licensing and regions required for geminin interaction. The C-terminal 377 residues of Cdt1 are required for licensing and the extreme C-terminus contains a domain that interacts with an Mcm(2,4,6,7) complex. Two regions of Cdt1 interact with geminin: one at the N-terminus, and one in the centre of the protein. Only the central region binds geminin tightly enough to successfully compete with full-length Cdt1 for geminin binding. This interaction requires a predicted coiled-coil domain that is conserved amongst metazoan Cdt1 homologues. Geminin forms a homodimer, with each dimer binding one molecule of Cdt1. Separation of the domains necessary for licensing activity from domains required for a strong interaction with geminin generated a construct, whose licensing activity was partially insensitive to geminin inhibition.

Animals↗
PubMed2004

Cdt1 downregulation by proteolysis and geminin inhibition prevents DNA re-replication in Xenopus.

In late mitosis and G1, Mcm2-7 are assembled onto replication origins to 'license' them for initiation. At other cell cycle stages, licensing is inhibited, thus ensuring that origins fire only once per cell cycle. Three additional factors--the origin recognition complex, Cdc6 and Cdt1--are required for origin licensing. We examine here how licensing is regulated in Xenopus egg extracts. We show that Cdt1 is downregulated late in the cell cycle by two different mechanisms: proteolysis, which occurs in part due to the activity of the anaphase-promoting complex (APC/C), and inhibition by a protein called geminin. If both these regulatory mechanisms are abrogated, extracts undergo uncontrolled re-licensing and re-replication. The extent of re-replication is limited by checkpoint kinases that are activated as a consequence of re-replication itself. These results allow us to build a comprehensive model of how re-replication of DNA is prevented in Xenopus, with Cdt1 regulation being the key feature. The results also explain the original experiments that led to the proposal of a replication licensing factor.

Animals↗
PubMed2004

Negative regulation of geminin by CDK-dependent ubiquitination controls replication licensing.

The replication licensing system ensures the precise duplication of chromosomal DNA in each cell cycle. In metazoans, a small protein called geminin plays a central role in negatively regulating licensing late in the cell cycle. Recent work using Xenopus egg extracts shows how geminin activity is downregulated on exit from metaphase in a process that requires mitotic cyclin-dependent kinases (CDKs). Geminin is polyubiquitinated by the Anaphase Promoting Complex, but instead of being proteolysed-the normal fate of polyubiquitinated proteins-much of the geminin is deubiquitinated, leaving it inactive. These results suggest a simple model for how precise chromosome duplication is ensured in the Xenopus model system.

Anaphase↗
PubMed2004

Non-proteolytic inactivation of geminin requires CDK-dependent ubiquitination.

In late mitosis and G1, a complex of the essential initiation proteins Mcm2-7 are assembled onto replication origins to 'license' them for initiation. At other times licensing is inhibited by cyclin-dependent kinases (CDKs) and geminin, thus ensuring that origins fire only once per cell cycle. Here we show that, paradoxically, CDKs are also required to inactivate geminin and activate the licensing system. On exit from metaphase in Xenopus laevis egg extracts, CDK-dependent activation of the anaphase-promoting complex (APC/C) results in the transient polyubiquitination of geminin. This ubiquitination triggers geminin inactivation without requiring ubiquitin-dependent proteolysis, and is essential for replication origins to become licensed. This reveals an unexpected role for CDKs and ubiquitination in activating chromosomal DNA replication.

Anaphase-Promoting Complex-Cyclosome↗
PubMed2002

Geminin becomes activated as an inhibitor of Cdt1/RLF-B following nuclear import.

During late mitosis and early interphase, origins of replication become "licensed" for DNA replication by loading Mcm2-7 complexes. Mcm2-7 complexes are removed from origins as replication forks initiate replication, thus preventing rereplication of DNA in a single cell cycle. Premature origin licensing is prevented in metaphase by the action of geminin, which binds and inhibits Cdt1/RLF-B, a protein that is required for the loading of Mcm2-7. Recombinant geminin that is added to Xenopus egg extracts is efficiently degraded upon exit from metaphase. Here, we show that recombinant and endogenous forms of Xenopus geminin behave differently from one another, such that a significant proportion of endogenous geminin escapes proteolysis upon exit from metaphase. During late mitosis and early G1, the surviving population of endogenous geminin does not associate with Cdt1/RLF-B and does not inhibit licensing. Following nuclear assembly, geminin is imported into nuclei and becomes reactivated to bind Cdt1/RLF-B. This reactivated geminin provides the major nucleoplasmic inhibitor of origin relicensing during late interphase. Since the initiation of replication at licensed origins depends on nuclear assembly, our results suggest an elegant and novel mechanism for preventing rereplication of DNA in a single cell cycle.

Active Transport, Cell Nucleus↗
PubMed2002

Repair of damaged DNA by Arabidopsis cell extract.

All living organisms have to protect the integrity of their genomes from a wide range of genotoxic stresses to which they are inevitably exposed. However, understanding of DNA repair in plants lags far behind such knowledge in bacteria, yeast, and mammals, partially as a result of the absence of efficient in vitro systems. Here, we report the experimental setup for an Arabidopsis in vitro repair synthesis assay. The repair of plasmid DNA treated with three different DNA-damaging agents, UV light, cisplatin, and methylene blue, after incubation with whole-cell extract was monitored. To validate the reliability of our assay, we analyzed the repair proficiency of plants depleted in AtRAD1 activity. The reduced repair of UV light- and cisplatin-damaged DNA confirmed the deficiency of these plants in nucleotide excision repair. Decreased repair of methylene blue-induced oxidative lesions, which are believed to be processed by the base excision repair machinery in mammalian cells, may indicate a possible involvement of AtRAD1 in the repair of oxidative damage. Differences in sensitivity to DNA polymerase inhibitors (aphidicolin and dideoxy TTP) between plant and human cell extracts were observed with this assay.

Aphidicolin↗