DNA replication licensing control and rereplication prevention

Eukaryotic DNA replication is tightly restricted to only once per cell cycle in order to maintain genome stability. Cells use multiple mechanisms to control the assembly of the prereplication complex (pre-RC), a process known as replication licensing. This review focuses on the regulation of replication licensing by posttranslational modifications of the licensing factors, including phosphorylation, ubiquitylation and acetylation. These modifications are critical in establishing the pre-RC complexes as well as preventing rereplication in each cell cycle. The relationship between rereplication and diseases, including cancer and virus infection, is discussed as well.     

Regulating the licensing of DNA replication origins in metazoa

Eukaryotic DNA replication is a highly conserved process; the proteins and sequence of events that replicate animal genomes are remarkably similar to those that replicate yeast genomes. Moreover, the assembly of prereplication complexes at DNA replication origins ('DNA licensing') is regulated in all eukaryotes so that no origin fires more than once in a single cell cycle. And yet there are significant differences between species both in the selection of replication origins and in the way in which these origins are licensed to operate. Moreover, these differences impart advantages to multicellular animals and plants that facilitate their development, such as better control over endoreduplication, flexibility in origin selection, and discrimination between quiescent and proliferative states.     

Cyclin A promotes S-phase entry via interaction with the replication licensing factor Mcm7

Cyclin A is known to promote S-phase entry in mammals, but its critical targets in this process have not been defined. We derived a novel human cyclin A mutant (CycA-C1), which can activate cyclin-dependent kinase but cannot promote S-phase entry, and isolated replication licensing factor Mcm7 as a factor that interacts with the wild-type cyclin A but not with the mutant. We demonstrated that human cyclin A and Mcm7 interact in the chromatin fraction. To address the physiological significance of the cyclin A-Mcm7 interaction, we isolated an Mcm7 mutant (Mcm7-3) that is capable of association with CycA-C1 and found that it can also suppress the deficiency of CycA-C1 in promoting S-phase entry. Finally, RNA interference experiments showed that the CycA-C1 mutant is defective for the endogenous cyclin A function in S-phase entry and that this defect can be suppressed by the Mcm7-3 mutant. Our findings demonstrate that interaction with Mcm7 is essential for the function of cyclin A in promoting S-phase entry.     

Human NOC3 is essential for DNA replication licensing in human cells

Noc3p (Nucleolar Complex-associated protein) is an essential protein in budding yeast DNA replication licensing. Noc3p mediates the loading of Cdc6p and MCM proteins onto replication origins during the M-to-G₁ transition by interacting with ORC (Origin Recognition Complex) and MCM (Minichromosome Maintenance) proteins. FAD24 (Factor for Adipocyte Differentiation, clone number 24), the human homolog of Noc3p (hNOC3), was previously reported to play roles in the regulation of DNA replication and proliferation in human cells. However, the role of hNOC3 in replication licensing was unclear. Here we report that hNOC3 physically interacts with multiple human pre-replicative complex (pre-RC) proteins and associates with known replication origins throughout the cell cycle. Moreover, knockdown of hNOC3 in HeLa cells abrogates the chromatin association of other pre-RC proteins including hCDC6 and hMCM, leading to DNA replication defects and eventual apoptosis in an abortive S-phase. In comparison, specific inhibition of the ribosome biogenesis pathway by preventing pre-rRNA synthesis, does not lead to any cell cycle or DNA replication defect or apoptosis in the same timeframe as the hNOC3 knockdown experiments. Our findings strongly suggest that hNOC3 plays an essential role in pre-RC formation and the initiation of DNA replication independent of its potential role in ribosome biogenesis in human cells.     

Life without kinase: cyclin E promotes DNA replication licensing and beyond

In a recent issue of Molecular Cell, Sicinski and colleagues report the surprising discovery that cyclin E promotes replication licensing and transformation independently of CDKs, resolving inconsistencies of inactivating cyclin E and CDK2 in the mouse and cells.     

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.     

Asynchronous DNA replication and origin licensing in the mouse one‐cell embryo

To prevent duplicate DNA synthesis, metazoan replication origins are licensed during G1. Only licensed origins can initiate replication, and the cytoplasm interacts with the nucleus to inhibit new licensing during S phase. DNA replication in the mammalian one‐cell embryo is unique because it occurs in two separate pronuclei within the same cytoplasm. Here, we first tested how long after activation the oocyte can continue to support licensing. Because sperm chromatin is licensed de novo after fertilization, the timing of sperm injection can be used to assay licensing initiation. To experimentally skip some of the steps of sperm decondensation, we injected mouse sperm halos into parthenogenetically activated oocytes. We found that de novo licensing was possible for up to 3 h after oocyte activation, and as early as 4 h before DNA replication began. We also found that the oocyte cytoplasm could support asynchronous initiation of DNA synthesis in the two pronuclei with a difference of at least 2 h. We next tested how tightly the oocyte cytoplasm regulates DNA synthesis by transferring paternal pronuclei from zygotes generated by intracytoplasmic sperm injection (ICSI) into parthenogenetically activated oocytes. The pronuclei from G1 phase zygotes transferred into S phase ooplasm were not induced to prematurely replicate and paternal pronuclei from S phase zygotes transferred into G phase ooplasm continued replication. These data suggest that the one‐cell embryo can be an important model for understanding the regulation of DNA synthesis. J. Cell. Biochem. 107: 214–223, 2009. © 2009 Wiley‐Liss, Inc.     

Bidentate and tridentate metal-ion coordination states within ternary complexes of RB69 DNA polymerase

Two divalent metal ions are required for primer‐extension catalyzed by DNA polymerases. One metal ion brings the 3′‐hydroxyl of the primer terminus and the α‐phosphorus atom of incoming dNTP together for bond formation so that the catalytically relevant conformation of the triphosphate tail of the dNTP is in an α,β,γ‐tridentate coordination complex with the second metal ion required for proper substrate alignment. A probable base selectivity mechanism derived from structural studies on Dpo4 suggests that the inability of mispaired dNTPs to form a substrate‐aligned, tridentate coordination complex could effectively cause the mispaired dNTPs to be rejected before catalysis. Nevertheless, we found that mispaired dNTPs can actually form a properly aligned tridentate coordination complex. However, complementary dNTPs occasionally form misaligned complexes with mutant RB69 DNA polymerases (RB69pols) that are not in a tridentate coordination state. Here, we report finding a β,γ‐bidentate coordination complex that contained the complementary dUpNpp opposite dA in the structure of a ternary complex formed by the wild type RB69pol at 1.88 Å resolution. Our observations suggest that several distinct metal‐ion coordination states can exist at the ground state in the polymerase active site and that base selectivity is unlikely to be based on metal‐ion coordination alone.     

The roles of Tyr391 and Tyr619 in RB69 DNA polymerase replication fidelity

In the family-B DNA polymerase of bacteriophage RB69, the conserved aromatic palm-subdomain residues Tyr391 and Tyr619 interact with the last primer-template base-pair. Tyr619 interacts via a water-mediated hydrogen bond with the phosphate of the terminal primer nucleotide. The main-chain amide of Tyr391 interacts with the corresponding template nucleotide. A hydrogen bond has been postulated between Tyr391 and the hydroxyl group of Tyr567, a residue that plays a key role in base discrimination. This hydrogen bond may be crucial for forcing an infrequent Tyr567 rotamer conformation and, when the bond is removed, may influence fidelity. We investigated the roles of these residues in replication fidelity in vivo employing phage T4 rII reversion assays and an rI forward assay. Tyr391 was replaced by Phe, Met and Ala, and Tyr619 by Phe. The Y391A mutant, reported previously to decrease polymerase affinity for incoming nucleotides, was unable to support DNA replication in vivo, so we used an in vitro fidelity assay. Tyr391F/M replacements affect fidelity only slightly, implying that the bond with Tyr567 is not essential for fidelity. The Y391A enzyme has no mutator phenotype in vitro. The Y619F mutant displays a complex profile of impacts on fidelity but has almost the same mutational spectrum as the parental enzyme. The Y619F mutant displays reduced DNA binding, processivity, and exonuclease activity on single-stranded DNA and double-stranded DNA substrates. The Y619F substitution would disrupt the hydrogen bond network at the primer terminus and may affect the alignment of the 3' primer terminus at the polymerase active site, slowing chemistry and overall DNA synthesis.     

体外PCR扩增和体内DNA复制有关问题的分析

体外PCR扩增和体内DNA复制是获得复制DNA的2种途径,它们都依据半保留复制的原理,但因其操作的环境不同,所要求的条件和具体的过程又有所不同。针对高中学生的特点对这2个过程所需要的条件、PCR扩增引物的设计、体内DNA复制冈崎片段的连接方式等进行了分析总结。     

Arabidopsis replication factor C4 is critical for DNA replication during the mitotic cell cycle

Replication factor C (RFC) is a conserved eukaryotic complex consisting of RFC1/2/3/4/5. It plays important roles in DNA replication and the cell cycle in yeast and fruit fly. However, it is not very clear how RFC subunits function in higher plants, except for the Arabidopsis (At) subunits AtRFC1 and AtRFC3. In this study, we investigated the functions of AtRFC4 and found that loss of function of AtRFC4 led to an early sporophyte lethality that initiated as early as the elongated zygote stage, all defective embryos arrested at the two‐ to four‐cell embryo proper stage, and the endosperm possessed six to eight free nuclei. Complementation of rfc4‐1/+ with AtRFC4 expression driven through the embryo‐specific DD45pro and ABI3pro or the endosperm‐specific FIS2pro could not completely restore the defective embryo or endosperm, whereas a combination of these three promoters in rfc4‐1/+ enabled the aborted ovules to develop into viable seeds. This suggests that AtRFC4 functions simultaneously in endosperm and embryo and that the proliferation of endosperm is critical for embryo maturation. Assays of DNA content in rfc4‐1/+ verified that DNA replication was disrupted in endosperm and embryo, resulting in blocked mitosis. Moreover, we observed a decreased proportion of late S‐phase and M‐phase cells in the rfc4‐1/–^{FIS2;DD45;ABI3pro::AtRFC4} seedlings, suggesting that incomplete DNA replication triggered cell cycle arrest in cells of the root apical meristem. Therefore, we conclude that AtRFC4 is a crucial gene for DNA replication.     

DNA replication in Physarum polycephalum: characterization of replication products in vivo.

Synchronous plasmodia of Physarum polycephalum in DNA synthesis were pulse-labelled with [oH]- thymidine for time periods of 15 seconds up to 9 minutes, or given a 30 seconds pulse followed by chase periods of 9 minutes up to 6 hours. Sedimentation analysis in alkaline sucrose gradients revealed at least five species of single stranded DNA14 molecules in the pulse experiments. Co-sedimentation of [14C]-labelled phage-DNA gave relative S-values of 5-7, 13-15, 23-25, 30 and 33-35 for these DNA molecules, all of which can be chased into DNA of higher molecular weight.     

Regulation of bacteriophage mu and mini-mu DNA replication in vivo

To study the regulation of bacteriophage Mu DNA's integrative-replication (transposition) during lytic growth in a cell containing both a Mu and a helper-dependent Mini-Mu (short, internally-deleted Mu genome), we placed "marker" genes (bla, lacZ) within either genome and then measured their encoded enzymes as indicators of the gene dosage. These results, corroborated using DNA-DNA hybridization, show that Mu and Mini-Mu DNA transposition is well regulated, requires both the Mu A and B gene products, and can be readily monitored by measuring beta-galactosidase and beta-lactamase expressed from the lacZ and bla genes, respectively.