Akari Yoshimura

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.

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.

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.

Budding yeast mms4 is epistatic with rad52 and the function of Mms4 can be replaced by a bacterial Holliday junction resolvase

MMS4 of Saccharomyces cerevisiae was originally identified as the gene responsible for one of the collection of methyl methanesulfonate (MMS)-sensitive mutants, mms4. Recently it was identified as a synthetic lethal gene with an SGS1 mutation. Epistatic analyses revealed that MMS4 is involved in a pathway leading to homologous recombination requiring Rad52 or in the recombination itself, in which SGS1 is also involved. MMS sensitivity of mms4 but not sgs1, was suppressed by introducing a bacterial Holliday junction (HJ) resolvase, RusA. The frequencies of spontaneously occurring unequal sister chromatid recombination (SCR) and loss of marker in the rDNA in haploid mms4 cells and interchromosomal recombination between heteroalleles in diploid mms4 cells were essentially the same as those of wild-type cells. Although UV- and MMS-induced interchromosomal recombination was defective in sgs1 diploid cells, hyper-induction of interchromosomal recombination was observed in diploid mms4 cells, indicating that the function of Mms4 is dispensable for this type of recombination.

Functional interactions between BLM and XRCC3 in the cell

Blooms syndrome (BS), which is caused by mutations in the BLM gene, is characterized by a predisposition to a wide variety of cancers. BS cells exhibit elevated frequencies of sister chromatid exchanges (SCEs), interchanges between homologous chromosomes (mitotic chiasmata), and sensitivity to several DNA-damaging agents. To address the mechanism that confers these phenotypes in BS cells, we characterize a series of double and triple mutants with mutations in BLM and in other genes involved in repair pathways. We found that XRCC3 activity generates substrates that cause the elevated SCE in blm cells and that BLM with DNA topoisomerase IIIα suppresses the formation of SCE. In addition, XRCC3 activity also generates the ultraviolet (UV)- and methyl methanesulfonate (MMS)–induced mitotic chiasmata. Moreover, disruption of XRCC3 suppresses MMS and UV sensitivity and the MMS- and UV-induced chromosomal aberrations of blm cells, indicating that BLM acts downstream of XRCC3.

Generation and characterization of cells that can be conditionally depleted of mitochondrial SOD2

Manganese-dependent superoxide dismutase (SOD2) serves as the primary defense against mitochondrial superoxide, and decreased SOD2 activity results in a range of pathologies. To investigate the events occurring soon after depletion of SOD2, we generated SOD2 gene knockout chicken DT40 cells complemented with a human SOD2 (hSOD2) cDNA, whose expression can be switched off by doxycycline (Dox). When SOD2 was depleted by the addition of Dox, the cells grew slightly slower and formed fewer colonies than cells expressing hSOD2. In addition, these cells showed a high sensitivity to paraquat, which produces superoxide, and died through apoptosis. In contrast to results obtained with mouse and DrosophilaSod2 mutants, we found no indication of an increase in DNA lesions due to depletion of SOD2.