human exonuclease 1 and BLM helicase
In a recent paper by Amitabh Nimonkar and colleagues, it was established that human BLM helicase, a member of the RecQ family, stimulates human exonuclease 1 (hExo1), a 5′ – 3′ double-stranded DNA exonuclease … “Stimulation of DNA resection by hExo1 is independent of BLM helicase activity and is, instead, mediated by an interaction between the 2 proteins. DNA ends resected by hExo1 and BLM are used by human Rad51, but not its yeast or bacterial counterparts, to promote homologous DNA pairing.”
Nimonkar AV, Oözsoy AZ, Genschel J, Modrich P, Kowalczykowski SC.
Human exonuclease 1 and BLM helicase interact to resect DNA and initiate DNA repair.
Proc Natl Acad Sci U S A. 2008 Oct 29. [Epub ahead of print]
Sarah Palin and the fruitfly
I know this is passe, but just for the record … on her criticism about unyielding pet projects, one example that popped into her head — fruitfly research … “I kid you not” … have a look on youtube … was she actually referring to Drosophila melanogaster? … read here … and here
WRN mini-review
In a recent issue of DNA Repair [7 (2008) 1776–1786], Julia Sidorova reviews the role of WRN in preserving DNA integrity during replication and propose that WRN can function in coordinating replication fork progression with replication stress-induced fork remodeling. She further discusses damage tolerance pathways, redundancy, and cooperation with other RecQ helicases.
Fig. 3. Possible scenarios of WRN function in coordinating fork progression with damage repair via control over daughter/daughter duplex expansion and/or half-life. (A) An unproductive daughter/daughter duplex with the 3′ overhang is unwound to redirect damage bypass towards translesion synthesis (TLS). (B) An extension of a daughter/daughter duplex leads to exposure of ssDNA regions of mother strands (for simplicity, only one of the strands is shown coated with RPA). Accumulation of RPA stimulates helicase activity of WRN to limit propagation of daughter/daughter duplex and restore an original fork conformation. (C) Lagging strand synthesis in the presence of a daughter/daughter duplex can lead to formation of long flaps. WRN can prevent their formation by limiting half-life of a daughter/daughter duplex, or stimulate FEN-1 to cleave such flaps once they are formed.
Back from Heidelberg
I’m back from Heidelberg. My original plan was to blog during the entire symposium, but that did not materialize. I will write more about the three days soon. In the meantime, here is an interesting paper on Sgs1 and BLM that recently came out on Genes and Development. Serge Gravel and colleagues studied 5′-to-3′ DSB resection by nucleases. They have observed that in the absence of exonuclease Exo1 activity, deletion or mutation of the Saccharomyces cerevisiae Sgs1 causes pronounced hypersensitivity to DSB-inducing agents. They revealed further that the mammalian Sgs1 ortholog, BLM, also functions in parallel with Exo1 to promote DSB resection, DSB signaling and resistance to DSB-generating agents.
Figure 4
Figure 4. BLM and EXO1 promote DNA DSB resection and associated events in human cells. (A) BLM and EXO1 deficiency impairs Camptothecin-induced RPA focus formation. U2OS cells were transfected with siRNAs directed against Luciferase (siCNTL), CtIP, BLM, EXO1, or a combination of BLM and EXO1, then 72 h later were mock-treated or treated with 1 µM Camptothecin for 1 h. Cells were next detergent-extracted and fixed, then foci for phosphorylated histone H2AX ({gamma}H2AX) and RPA2 were visualized by indirect immunofluorescence. More than 100 cells were counted for each sample and the percentages of cells exhibiting both {gamma}H2AX and RPA foci was determined. Data represent the mean ± SEM from three independent experiments. All counting was done blind. (B) BLM and EXO1 promote DSB signaling. Extracts of mock-treated or Camptothecin-treated cells depleted for the indicated factors were analyzed by Western blotting with the indicated antibodies. Endogenous EXO1 levels were too low to allow detection with anti-EXO1 antisera and so verification of EXO1 siRNA depletion was done with cells stably expressing a GFP-Exo1 construct (see Supplemental Fig. S4). (C) RPA Ser-4 and Ser-8 phosphorylation (RPApS4/S8) is compromised by BLM and EXO1 depletion. CtIP, BLM, or EXO1 were depleted and cells were treated as in A, followed by analysis by indirect immunofluorescence with the indicated antibodies. More than 100 cells were counted for each sample, and data represent the mean ± SEM from three independent experiments. Counting was done blind. (D) Codepletion or BLM and EXO1 yields Camptothecin hypersensitivity. Seventy-two hours following transfection with the indicated siRNAs, U2OS cells were treated with Camptothecin for 1 h, and cell survival was determined by colony formation. Data represent the mean ± SEM from three independent experiments.
sports when you’re old
I saw a video (Super Geezballs by Lance Brown) recently showing cartoon images of old people engaged in a variety of sports, including skateboarding, acrobatics, golf, tennis, skating, skydiving. It said something like … “what the spirit would do, if the body were able” …
rDNA and recombination
In a mini-review by Ellen Tsang and Anthony Carr, recombination is highly regulated in the rDNA. Repetitive sequences such as rDNA provide good substrates for homologous recombination, particualrly if replication forks collapse here. Several studies have shown that replication fork barrier (RFB)-dependent and -independent replication fork arrest, as well as replication-independent DNA metabolism, can induce recombination in the rDNA. Some of the molecules involved in regulating recombination include Fob1, Sir2, topoisomerase, RNA polymerase I, and HOT1.
They concluded by stating:
“… high levels of rDNA recombination acts to maintain sequence uniformity … it should be noted that the outcomes of recombination within the rDNA are very different to those of recombination at other repeat units within the genome … the specialised nature of the rDNA metabolism suggests that observations made at the rDNA locus may not always be applicable to the wider genome … fork arrest at a replication barrier only generates the potential for recombination; whether recombination occurs or not, and through which pathways, remains the responsibility of other local factors, such as the binding of regulatory protein complexes and sister chromatid cohesion …”
Tsang E, Carr AM.
Replication fork arrest, recombination and the maintenance of ribosomal DNA stability.
DNA Repair (Amst). 2008 Oct 1;7(10):1613-23. Epub 2008 Jul 29.
Fig. 2. Model for the regulation of recombination outcomes in the rDNA. Heavy arrows represent individual rDNA units; those shown in blue are equivalent units on sister chromatids. The open circle denotes an active rARS; the red bar denotes an active RFB. Leftward-moving replication forks arrest at the RFB. A subset is thought to collapse, requiring recombination to restart replication. Under normal circumstances, sister chromatids are held together by cohesin (grey rings), forcing the broken end to invade the equivalent unit to give equal sister chromatid exchange and rDNA stability (A). In cases where cohesin is dissociated from the rDNA, the broken end can invade either an upstream (B) or downstream (C) unit, leading to expansion or contraction of the rDNA array, respectively [17].
PDF (469 K)[17] T. Kobayashi, T. Horiuchi, P. Tongaonkar, L. Vu and M. Nomura, SIR2 regulates recombination between different rDNA repeats, but not recombination within individual rRNA genes in yeast, Cell 117 (2004), pp. 441–453.
mitochondrial DNA subunits
Rajindar Sohal and colleagues recently made a study comparing the expression of mitochondrial DNA encoded subunits of cytochrome c oxidase (CcO) in Drosophila melanogaster. They compared mitochondrial proteins from 15-, 25-, 35-, 47- and 60-day-old flies. Subunits I, II and III constitute the catalytic functional core of CcO. They found out that there are differential losses in subunits I, II and III. They wrote:
“Subunits II and III decreased with age by up to 43% and 75%, respectively, whereas the decrease in subunit I was only 15% … This could theoretically be explained, at least in part, by mitochondrial DNA deletions, which affect some gene sequences more frequently than others, and which are known to accumulate as a function of age in Drosophila.”
Sohal RS, Toroser D, Brégère C, Mockett RJ, Orr WC.
Age-related decrease in expression of mitochondrial DNA encoded subunits of cytochrome c oxidase in Drosophila melanogaster.
Mech Ageing Dev. 2008 Sep;129(9):558-61. Epub 2008 Apr 22.
Fig. 1. Effect of age on the abundance of cytochrome c oxidase subunits in Drosophila melanogaster. Proteins (10 μg/lane) from flight muscle mitochondria were separated by discontinuous 10% SDS-PAGE and stained with Coomassie Blue (A), or probed with anti-complex Vα, anti-porin (B) and anti-CcO subunits I, II, III and VIb (C), Results presented in panel D are means of at least three samples, obtained from two independent mitochondrial preparations. For purposes of clarity, error bars are not shown (the coefficient of variation was <20% for all points).
eat heavy atoms, live longer
What kind of diet helps to live longer? In a review paper by Michael Heinrich and Jose Prieto present a comprehensive assessment of how food has changed over time, how is has affect our health.
“Increased life expectancy has resulted in an increase in the quest for diets which allow for a healthy ageing.”
From the Mediterranean diet of oil and wild plants, the PUFAS in the Inuit diet, phytoestrogens in the Asian diet, milk, snails, insects, algae, to vegetarianism, all sorts of diet were reviewed.
Apparently, it has very recently been shown that a 10% extension of the lifespan in Caenorhabditis elegans worms can be achieved by feeding them with nutrients reinforced with natural “heavy” isotopes. This is due to the fact that the bodily constituents and DNA produced from isotopically-reinforced nutrients are more resistant to free radical attack (believed to contribute to ageing).
