The prokaryotic genomes, for which complete nucleotide sequences are available, always contain at least one RNase H gene, indicating that RNase H is ubiquitous in all prokaryotic cells. Coupled with its unique substrate specificity, the enzyme has been expected to play crucial roles in the biochemical processes associated with DNA replication, gene expression and DNA repair. The physiological role of prokaryotic RNases H, especially of type 1 RNases H, has been extensively studied using Escherichia coli strains that are defective in RNase HI activity or overproduce RNase HI. However, it is not fully understood yet. By contrast, significant progress has been made in this decade in identifying novel RNases H with respect to their biochemical properties and structures, and elucidating catalytic mechanism and substrate recognition mechanism of RNase H. We review the results of these studies.

Glutamate, the major excitatory neurotransmitter in the brain, activates receptors coupled to membrane depolarization and Ca 2؉ influx that mediates functional responses of neurons including processes such as learning and memory. Here we show that reversible nuclear oxidative DNA damage occurs in cerebral cortical neurons in response to transient glutamate receptor activation using non-toxic physiological levels of glutamate. This DNA damage was prevented by intracellular Ca 2؉ chelation, the mitochondrial superoxide dismutase mimetic MnTMPyP (Mn-5,10,15,20-tetra(4-pyridyl)-21H,23H-porphine chloride tetrakis(methochloride)), and blockade of the permeability transition pore. The repair of glutamate-induced DNA damage was associated with increased DNA repair activity and increased mRNA and protein levels of apurinic endonuclease 1 (APE1 which mediate long lasting changes in neuronal structure and function (2-5). Glutamate receptor activation also stimulates an increase in mitochondrial respiration (electron transport) to generate the ATP necessary to drive the activity of ion-motive ATPases that restore ion gradients across cellular membranes (6). Mitochondrial Ca 2ϩ uptake and increased mitochondrial respiration can result in production of the damaging free radical superoxide (7, 8), as well as mitochondrial membrane permeability changes that trigger cell death, a process called excitotoxicity (9, 10).Damage to DNA in neurons occurs early during excitotoxicity (11, 12) and may be a pivotal event in cell death because selective inhibition or knockdown of the DNA damage response proteins p53 (13, 14) and PARP-1 (15, 16) can prevent glutamate-induced neuronal death. Ca 2ϩ and mitochondriaderived superoxide are believed to play key roles in glutamateinduced DNA damage and cell death because PARP-1 activation is mediated by Ca 2ϩ and mitochondrial reactive oxygen species (17), and because mitochondrial Mn-SOD and exogenous antioxidants protect neurons against excitotoxicity (18 -20). However, it is not known if oxidative lesions to nuclear DNA occur in response to non-pathological subtoxic levels of glutamate receptor activation, nor is it known if and how neurons might respond to such glutamate-induced DNA damage.Base excision repair (BER) is the primary DNA repair pathway for removal of small base modifications such as alkylation, deamination, and oxidation (21,22). This process occurs both in the nucleus and in mitochondria. By far most information on the molecular mechanisms of DNA damage and repair comes from studies of non-neuronal cells, and the extent of DNA damage and repair in neurons under physiological and pathological conditions is largely unknown (23

DNA decatenation mediated by Topoisomerase II is required to separate the interlinked sister chromatids post-replication. SGS1, a yeast homolog of the human RecQ family of helicases interacts with Topoisomerase II and plays a role in chromosome segregation, but this functional interaction has yet to be identified in higher organisms. Here, we report a physical and functional interaction of Topoisomerase IIα with RECQL5, one of five mammalian RecQ helicases, during DNA replication. Direct interaction of RECQL5 with Topoisomerase IIα stimulates the decatenation activity of Topoisomerase IIα. Consistent with these observations, RECQL5 co-localizes with Topoisomerase IIα during S-phase of the cell cycle. Moreover, cells with stable depletions of RECQL5 display a slow proliferation rate, a G2/M cell cycle arrest and late S-phase cycling defects. Metaphase spreads generated from RECQL5-depleted cells exhibit undercondensed and entangled chromosomes. Further, RECQL5-depleted cells activate a G2/M checkpoint and undergo apoptosis. These phenotypes are similar to those observed when Topoisomerase II catalytic activity is inhibited. These results reveal an important role for RECQL5 in the maintenance of genomic stability and a new insight into the decatenation process.

Neurons are terminally differentiated cells with a high rate of metabolism and multiple biological properties distinct from their undifferentiated precursors. Previous studies showed that nucleotide excision DNA repair is down-regulated in post-mitotic muscle cells and neurons. Here, we characterize DNA damage susceptibility and base excision DNA repair (BER) capacity in undifferentiated and differentiated human neural cells. The results show that undifferentiated human SH-SY5Y neuroblastoma cells are less sensitive to oxidative damage than their differentiated counterparts, in part due to having robust BER capacity, which is heavily attenuated in the post-mitotic neurons. The reduction in BER activity in the differentiated cells correlates with diminished protein levels of key long patch BER components FEN-1, PCNA and Ligase I. Thus, due to their higher BER capacity, proliferative neural progenitor cells are more efficient at repairing DNA damage compared to their neuronally differentiated progeny.

We demonstrate 50-Gb/s direct modulation by using 1.3-μm distributed-feedback lasers with a ridge waveguide structure. We employed InGaAlAs material for a multiple-quantum well to obtain a low damping factor K, and fabricated a ridge waveguide structure buried in benzocyclobutene to realize a structure with a low parasitic capacitance. In addition, to obtain high maximum frequency relaxation oscillations fr, we designed the cavity length L), and achieved a 3-dB-down frequency bandwidth of 34 GHz. We realized 50-Gb/s clear eye openings with a back-toback configuration, and achieved a mean output power of over 5.0 dBm, and a dynamic extinction ratio of 4.5 dB. We measured the 50-Gb/s transmission characteristics, and obtained clear eye openings for transmissions over 20-, 40-, and 60-km single-mode fibers (SMF). We also measured the bit-error-rate performance, and obtained an error-free operation and a power penalty of less than 0.5 dB after a 10-km SMF transmission.

Ribonuclease (RNase) HI from the psychrotrophic bacterium Shewanella oneidensis MR-1 was overproduced in Escherichia coli, purified, and structurally and biochemically characterized. The amino acid sequence of MR-1 RNase HI is 67% identical to that of E. coli RNase HI. The crystal structure of MR-1 RNase HI determined at 2.0 A resolution was highly similar to that of E. coli RNase HI, except that the number of intramolecular ion pairs and the fraction of polar surface area of MR-1 RNase HI were reduced compared to those of E. coli RNase HI. The enzymatic properties of MR-1 RNase HI were similar to those of E. coli RNase HI. However, MR-1 RNase HI was much less stable than E. coli RNase HI. The stability of MR-1 RNase HI against heat inactivation was lower than that of E. coli RNase HI by 19 degrees C. The conformational stability of MR-1 RNase HI was thermodynamically analyzed by monitoring the CD values at 220 nm. MR-1 RNase HI was less stable than E. coli RNase HI by 22.4 degrees C in Tm and 12.5 kJ/mol in DeltaG(H2O). The thermodynamic stability curve of MR-1 RNase HI was characterized by a downward shift and increased curvature, which results in an increased DeltaCp value, compared to that of E. coli RNase HI. Site-directed mutagenesis studies suggest that the difference in the number of intramolecular ion pairs partly accounts for the difference in stability between MR-1 and E. coli RNases HI.

Five human RecQ helicases are involved in genome maintenance. RECQL5, one of the important members of this helicase family, is involved in DNA single-strand break repair and base excision DNA repair.

Humans have five RecQ helicases, whereas simpler organisms have only one. Little is known about whether and how these RecQ helicases co-operate and/or complement each other in response to cellular stress. Here we show that RECQL5 associates longer at laser-induced DNA double-strand breaks in the absence of Werner syndrome (WRN) protein, and that it interacts physically and functionally with WRN both in vivo and in vitro. RECQL5 co-operates with WRN on synthetic stalled replication fork-like structures and stimulates its helicase activity on DNA fork duplexes. Both RECQL5 and WRN re-localize from the nucleolus into the nucleus after replicative stress and significantly associate with each other during S-phase. Further, we show that RECQL5 is essential for cell survival in the absence of WRN. Loss of both RECQL5 and WRN severely compromises DNA replication, accumulates genomic instability and ultimately leads to cell death. Collectively, our results indicate that RECQL5 plays both co-operative and complementary roles with WRN. This is an early demonstration of a significant functional interplay and a novel synthetic lethal interaction among the human RecQ helicases.