Mobile D-loops are a preferred substrate for the Bloom's syndrome helicase

The Bloom's syndrome helicase, BLM, is a member of the highly conserved RecQ family, and possesses both DNA unwinding and DNA strand annealing activities. BLM also promotes branch migration of Holliday junctions. One role for BLM is to act in conjunction with topoisomerase IIIα to process homologous recombination (HR) intermediates containing a double Holliday junction by a process termed dissolution. However, several lines of evidence suggest that BLM may also act early in one or more of the recombination pathways to eliminate illegitimate or aberrantly paired DNA joint molecules. We have investigated whether BLM can disrupt DNA displacement loops (D-loops), which represent the initial strand invasion step of HR. We show that mobile D-loops created by the RecA recombinase are a highly preferred substrate for BLM with the invading strand being displaced from the duplex. We have identified structural features of the D-loop that determine the efficiency with which BLM promotes D-loop dissociation. We discuss these results in the context of models for the role of BLM as an ‘anti-recombinase’

Inflammation-induced DNA damage and damage-induced inflammation: a vicious cycle

Inflammation is the ultimate response to the constant challenges of the immune system by microbes, irritants or injury. The inflammatory cascade initiates with the recognition of microorganism-derived pathogen associated molecular patterns (PAMPs) and host cell-derived damage associated molecular patterns (DAMPs) by the pattern recognition receptors (PRRs). DNA as a molecular PAMP or DAMP is sensed directly or via specific binding proteins to instigate pro-inflammatory response. Some of these DNA binding proteins also participate in canonical DNA repair pathways and recognise damaged DNA to initiate DNA damage response. In this review we aim to capture the essence of the complex interplay between DNA damage response and the pro-inflammatory signalling through representative examples

ATRX dysfunction Induces replication defects in primary mouse cells

The chromatin remodeling protein ATRX, which targets tandem repetitive DNA, has been shown to be required for expression of the alpha globin genes, for proliferation of a variety of cellular progenitors, for chromosome congression and for the maintenance of telomeres. Mutations in ATRX have recently been identified in tumours which maintain their telomeres by a telomerase independent pathway involving homologous recombination thought to be triggered by DNA damage. It is as yet unknown whether there is a central underlying mechanism associated with ATRX dysfunction which can explain the numerous cellular phenomena observed. There is, however, growing evidence for its role in the replication of various repetitive DNA templates which are thought to have a propensity to form secondary structures. Using a mouse knockout model we demonstrate that ATRX plays a direct role in facilitating DNA replication. Ablation of ATRX alone, although leading to a DNA damage response at telomeres, is not sufficient to trigger the alternative lengthening of telomere pathway in mouse embryonic stem cells

RecQ helicases: suppressors of tumorigenesis and premature aging.

The RecQ helicases represent a subfamily of DNA helicases that are highly conserved in evolution. Loss of RecQ helicase function leads to a breakdown in the maintenance of genome integrity, in particular hyper-recombination. Germ-line defects in three of the five known human RecQ helicases give rise to defined genetic disorders associated with cancer predisposition and/or premature aging. These are Bloom's syndrome, Werner's syndrome and Rothmund-Thomson syndrome, which are caused by defects in the genes BLM, WRN and RECQ4 respectively. Here we review the properties of RecQ helicases in organisms from bacteria to humans, with an emphasis on the biochemical functions of these enzymes and the range of protein partners that they operate with. We will discuss models in which RecQ helicases are required to protect against replication fork demise, either through prevention of fork breakdown or restoration of productive DNA synthesis

A simple and efficient method for PCR amplifiable DNA extraction from ancient bones

A simple and effective modified ethanol precipitation-based protocol is described for the preparation of DNA from ancient human bones. This method is fast and requires neither hazardous chemicals nor special devices. After the powdering and incubating of the bone samples Dextran Blue was added as a carrier for removing the PCR inhibitors with selective ethanol precipitation. This method could eliminate the time-consuming separate decalcification step, dialysis, application of centrifugation-driven microconcentrators and the second consecutive PCR amplification. The efficiency of this procedure was demonstrated on ten 500–1200-year-old human bones from four different Hungarian burial sites. A mitochondrial specific primer pair was used to obtain sequence information from the purified ancient DNA. The PCR amplification, after our DNA extraction protocol, was successful from each of the 10 bone samples investigated. The results demonstrate that extraction of DNA from ancient bone samples with this new approach increases the success rate of PCR amplification

Comparison of the efficiency of unwinding of different substrates by BLM

<p><b>Copyright information:</b></p><p>Taken from "Mobile D-loops are a preferred substrate for the Bloom's syndrome helicase"</p><p>Nucleic Acids Research 2006;34(8):2269-2279.</p><p>Published online 2 May 2006</p><p>PMCID:PMC1456333.</p><p>© The Author 2006. Published by Oxford University Press. All rights reserved</p> BLM enzyme-concentration dependent unwinding of the pDL3 mobile D-loop (upper panel), the X12 four-way junction (middle panel) and G4 (lower panel) substrates. The enzyme-concentration was varied between 1–40 nM as indicated above by the triangle. Reactions lacking BLM are denoted by ‘−’ above. The flame symbol denotes heat-denatured DNA. The substrates and the products of unwinding are shown on the left of each panel. () Quantification of the data from (). Blue triangle, green diamond and red square symbols depict the pDL3, four-way junction and G4 DNA substrates, respectively. Analysis of the concentration-dependence curve indicated that 50% unwinding of pDL3, four-way junction and G4 DNA is achieved at 7.38, 8.97 and 5.84 nM, respectively. At high concentrations of BLM, the strand annealing activity of the enzyme becomes dominant over the unwinding activity, resulting in a diminution in the level of unwound product for the four-way junction substrate [middle panel of (a), green triangles and dotted line of (b)]. The last two concentration points (30 and 40 nM) were, therefore, omitted from the non-linear regression analysis and for determination of the 50% unwinding concentration (see Materials and Methods)

Kinetics of unwinding of oligonucleotide-based D-loops in a time-course experiment

<p><b>Copyright information:</b></p><p>Taken from "Mobile D-loops are a preferred substrate for the Bloom's syndrome helicase"</p><p>Nucleic Acids Research 2006;34(8):2269-2279.</p><p>Published online 2 May 2006</p><p>PMCID:PMC1456333.</p><p>© The Author 2006. Published by Oxford University Press. All rights reserved</p> Unwinding was carried out in the presence of 0.64 nM BLM for the period of time indicated above the lanes. The position of the substrate and fully unwound single-stranded oligonucleotide products, together with reaction intermediates A and B, are shown on each side. The flame symbol denotes heat-denatured DNA