Shusuke Tada

Structural basis for inhibition of the replication licensing factor Cdt1 by geminin

To maintain chromosome stability in eukaryotic cells, replication origins must be licensed by loading mini-chromosome maintenance (MCM2–7) complexes once and only once per cell cycle. This licensing control is achieved through the activities of geminin and cyclin-dependent kinases. Geminin binds tightly to Cdt1, an essential component of the replication licensing system, and prevents the inappropriate reinitiation of replication on an already fired origin. The inhibitory effect of geminin is thought to prevent the interaction between Cdt1 and the MCM helicase. Here we describe the crystal structure of the mouse geminin–Cdt1 complex using tGeminin (residues 79–157, truncated geminin) and tCdt1 (residues 172–368, truncated Cdt1). The amino-terminal region of a coiled-coil dimer of tGeminin interacts with both N-terminal and carboxy-terminal parts of tCdt1. The primary interface relies on the steric complementarity between the tGeminin dimer and the hydrophobic face of the two short N-terminal helices of tCdt1 and, in particular, Pro 181, Ala 182, Tyr 183, Phe 186 and Leu 189. The crystal structure, in conjunction with our biochemical data, indicates that the N-terminal region of tGeminin might be required to anchor tCdt1, and the C-terminal region of tGeminin prevents access of the MCM complex to tCdt1 through steric hindrance.

Functional relation among RecQ family helicases RecQL1, RecQL5, and BLM in cell growth and sister chromatid exchange formation.

ABSTRACT Human RECQL1 and RECQL5 belong to the RecQ family that includes Bloom syndrome, Werner syndrome, and Rothmund-Thomson syndrome causative genes. Cells derived from individuals suffering from these syndromes show significant levels of genomic instability. However, neither RECQL1 nor RECQL5 has been related to a disease, and nothing is known about the functions of RecQL1 and RecQL5. We generated here RECQL1−/− , RECQL5−/− , RECQL1−/− /RECQL5−/− , RECQL1−/− /BLM−/− , and RECQL5−/− /BLM−/− cells from chicken B-lymphocyte line DT40 cells. Although BLM−/− DT40 cells showed a slow-growth phenotype, a higher sensitivity to methyl methanesulfonate than the wild type, and an ∼10-fold increase in the frequency of sister chromatid exchange (SCE) compared to wild-type cells, RECQL1−/− , RECQL5−/− , and RECQL1−/− /RECQL5−/− cells showed no significant difference from the wild-type cells in growth, sensitivity to DNA-damaging agents, and the frequency of SCE. However, both RECQL1−/− /BLM−/− and RECQL5−/− /BLM−/− cells grew more slowly than BLM−/− cells because of the increase in the population of dead cells, indicating that RecQL1 and RecQL5 are somehow involved in cell viability under the BLM function-impaired condition. Surprisingly, RECQL5−/− /BLM−/− cells showed a higher frequency of SCE than BLM−/− cells, indicating that RecQL5 suppresses SCE under the BLM function-impaired condition.

The Histone Chaperone Facilitates Chromatin Transcription (FACT) Protein Maintains Normal Replication Fork Rates

Ordered nucleosome disassembly and reassembly are required for eukaryotic DNA replication. The facilitates chromatin transcription (FACT) complex, a histone chaperone comprising Spt16 and SSRP1, is involved in DNA replication as well as transcription. FACT associates with the MCM helicase, which is involved in DNA replication initiation and elongation. Although the FACT-MCM complex is reported to regulate DNA replication initiation, its functional role in DNA replication elongation remains elusive. To elucidate the functional role of FACT in replication fork progression during DNA elongation in the cells, we generated and analyzed conditional SSRP1 gene knock-out chicken (Gallus gallus) DT40 cells. SSRP1-depleted cells ceased to grow and exhibited a delay in S-phase cell cycle progression, although SSRP1 depletion did not affect the level of chromatin-bound DNA polymerase α or nucleosome reassembly on daughter strands. The tracking length of newly synthesized DNA, but not origin firing, was reduced in SSRP1-depleted cells, suggesting that the S-phase cell cycle delay is mainly due to the inhibition of replication fork progression rather than to defects in the initiation of DNA replication in these cells. We discuss the mechanisms of how FACT promotes replication fork progression in the cells.

Licensing for DNA replication requires a strict sequential assembly of Cdc6 and Cdt1 onto chromatin in Xenopus egg extracts

Replication origins are licensed for a single initiation event by the loading of Mcm2-7 proteins during late mitosis and G1. Sequential associations of origin recognition complex, Cdc6 and Mcm2-7 are essential for completion of the licensing. Although Cdt1 also binds to the chromatin when the licensing reaction takes place, whether the binding is a requirement for Cdt1 to function is unclear. To analyze the relevance of the chromatin association of Cdt1, we carried out chromatin transfer experiments using either immunodepleted Xenopus egg extracts or purified proteins. Licensing assay and immunoblotting analyses indicated that Cdt1 could only license DNA replication and load Mcm2-7 onto DNA when it binds to chromatin that has already associated with Cdc6. These results provide evidence supporting that Cdc6 and Cdt1 must bind to chromatin in a strict order for DNA licensing to occur.

Bloom Helicase and DNA Topoisomerase IIIα Are Involved in the Dissolution of Sister Chromatids

ABSTRACT Blooms syndrome (BS) is an autosomal disorder characterized by predisposition to a wide variety of cancers. The gene product whose mutation leads to BS is the RecQ family helicase BLM, which forms a complex with DNA topoisomerase IIIα (Top3α). However, the physiological relevance of the interaction between BLM and Top3α within the cell remains unclear. We show here that Top3α depletion causes accumulation of cells in G2 phase, enlargement of nuclei, and chromosome gaps and breaks that occur at the same position in sister chromatids. The transition from metaphase to anaphase is also inhibited. All of these phenomena except cell lethality are suppressed by BLM gene disruption. Taken together with the biochemical properties of BLM and Top3α, these data indicate that BLM and Top3α execute the dissolution of sister chromatids.

The N-terminal region of RECQL4 lacking the helicase domain is both essential and sufficient for the viability of vertebrate cells: Role of the N-terminal region of RECQL4 in cells

Rothmund-Thomson syndrome (RTS) is a rare genetic disorder characterized by premature aging, developmental abnormalities, and a predisposition to cancer. RTS is caused by mutations in the RECQL4 gene, which encodes one of the five human RecQ helicases. To identify the cellular functions of RECQL4, we generated a chicken DT40 cell line in which RECQL4 expression could be turned off by doxycycline (Dox). Upon exposure to Dox, cells stopped growing and underwent apoptosis. The cells could be rescued by expression of the N-terminal region of RECQL4 (amino acids 1-496), which lacks the helicase domain and has sequence similarity to yeast Sld2, which plays an essential function in the initiation of DNA replication in Saccharomyces cerevisiae. Smaller fragments of the N-terminal region of RECQL4 did not rescue the cells from lethality. RECQL4 gene knockout cells complemented with RECQL4 (1-496) showed relatively high sensitivity to DNA damaging agents that induce double strand breaks and cross-links, suggesting that the C-terminal region including the helicase domain of RECQL4 is involved in the repair of certain types of DNA lesions.

Rad52 sumoylation and its involvement in the efficient induction of homologous recombination

The protein Rad52 is a key player in various types of homologous recombination and is essential to maintenance of genomic integrity. Although evidence indicates that Rad52 is modified by SUMO, the physiological relevance of this sumoylation remains unclear. Here, we identify the conditions under which Rad52 sumoylation is induced, and clarify the role of this modification in homologous recombination. Oligomerization of Rad52 was a prerequisite for sumoylation, and the modification occurred in the cell proceeding S phase being exposed to the DNA-damaging agent methyl methanesulfonate (MMS). Following exposure to MMS, sumoylated Rad52 accumulated in rad51 cells, but not in the recombination-related gene mutants, rad54, rad55, rad59, sgs1, or srs2. The accumulation of sumoylated Rad52 was suppressed in rad51 cells expressing Rad51-K191R, an ATPase-defective protein presumed to be recruited to ssDNA. Although the sumoylation defective mutant rad52-3KR (K10R/K11R/K220R) showed no defect in mating-type switching, which did not lead to Rad52 sumoylation in wild-type cells, the mutant did demonstrate a partial defect in MMS-induced interchromosomal homologous recombination.

KU70/80, DNA-PKcs, and Artemis are essential for the rapid induction of apoptosis after massive DSB formation

KU70(-/-) and DNA-PKcs(-/-/-)chicken DT40 cells are reportedly highly sensitive to the DNA topoisomerase II inhibitor etoposide. Here we report that KU70 and DNA-PKcs unexpectedly function together during the induction of apoptosis after exposure to high levels of etoposide. In the presence of 100 microM etoposide, apoptosis was induced within 1 h in wild type DT40 cells but not in KU70(-/-) and DNA-PKcs(-/-/-) cells. In addition, the DNA-PK inhibitors NU7026 and wortmannin, as well as the caspase inhibitor Z-VAD-FMK, inhibited etoposide-induced apoptosis in wild type cells. Although Artemis(-/-) cells also showed defects in the etoposide-induced apoptosis, the other mutants defective in nonhomologous end-joining (NHEJ), LIG4(-/-), XRCC4(-), and XLF(-/-) cells were capable to induce apoptosis. When cells were treated with high doses of etoposide, the chromatin binding of DNA-PKcs was impaired by deletion of KU70 but not of Artemis, suggesting that KU70 acts upstream of DNA-PKcs and Artemis acts downstream of DNA-PKcs in the apoptotic pathway like the NHEJ pathway. These results suggest that the proteins involved in the early stage of NHEJ pathway including Artemis but not the downstream factors decide the cell fate by selecting apoptosis or DNA repair according to the degree of DNA damage.

SOD1 Is Essential for the Viability of DT40 Cells and Nuclear SOD1 Functions as a Guardian of Genomic DNA

Reactive oxygen species (ROSs) are produced during normal cellular metabolism, particularly by respiration in mitochondria, and these ROSs are considered to cause oxidative damage to macromolecules, including DNA. In our previous paper, we found no indication that depletion of mitochondrial superoxide dismutase, SOD2, resulted in an increase in DNA damage. In this paper, we examined SOD1, which is distributed in the cytoplasm, nucleus, and mitochondrial intermembrane space. We generated conditional SOD1 knockout cells from chicken DT40 cells and analyzed their phenotypes. The results revealed that SOD1 was essential for viability and that depletion of SOD1, especially nuclear SOD1, increased sister chromatid exchange (SCE) frequency, suggesting that superoxide is generated in or near the nucleus and that nuclear SOD1 functions as a guardian of the genome. Furthermore, we found that ascorbic acid could offset the defects caused by SOD1 depletion, including cell lethality and increases in SCE frequency and apurinic/apyrimidinic sites.

Structure of the Cdt1 C-terminal domain: Conservation of the winged helix fold in replication licensing factors

In eukaryotic replication licensing, Cdt1 plays a key role by recruiting the MCM2‐7 complex onto the origin of chromosome. The C‐terminal domain of mouse Cdt1 (mCdt1C), the most conserved region in Cdt1, is essential for licensing and directly interacts with the MCM2‐7 complex. We have determined the structures of mCdt1CS (mCdt1C_small; residues 452 to 557) and mCdt1CL (mCdt1C_large; residues 420 to 557) using X‐ray crystallography and solution NMR spectroscopy, respectively. While the N‐terminal 31 residues of mCdt1CL form a flexible loop with a short helix near the middle, the rest of mCdt1C folds into a winged helix structure. Together with the middle domain of mouse Cdt1 (mCdt1M, residues 172–368), this study reveals that Cdt1 is formed with a tandem repeat of the winged helix domain. The winged helix fold is also conserved in other licensing factors including archaeal ORC and Cdc6, which supports an idea that these replication initiators may have evolved from a common ancestor. Based on the structure of mCdt1C, in conjunction with the biochemical analysis, we propose a binding site for the MCM complex within the mCdt1C.