RECQL5 cooperates with Topoisomerase II alpha in DNA decatenation and cell cycle progression.

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.

The human RecQ helicases BLM and RECQL4 cooperate to preserve genome stability.

Bacteria and yeast possess one RecQ helicase homolog whereas humans contain five RecQ helicases, all of which are important in preserving genome stability. Three of these, BLM, WRN and RECQL4, are mutated in human diseases manifesting in premature aging and cancer. We are interested in determining to which extent these RecQ helicases function cooperatively. Here, we report a novel physical and functional interaction between BLM and RECQL4. Both BLM and RECQL4 interact in vivo and in vitro. We have mapped the BLM interacting site to the N-terminus of RECQL4, comprising amino acids 361-478, and the region of BLM encompassing amino acids 1-902 interacts with RECQL4. RECQL4 specifically stimulates BLM helicase activity on DNA fork substrates in vitro. The in vivo interaction between RECQL4 and BLM is enhanced during the S-phase of the cell cycle, and after treatment with ionizing radiation. The retention of RECQL4 at DNA double-strand breaks is shortened in BLM-deficient cells. Further, depletion of RECQL4 in BLM-deficient cells leads to reduced proliferative capacity and an increased frequency of sister chromatid exchanges. Together, our results suggest that BLM and RECQL4 have coordinated activities that promote genome stability.

RECQL4, the protein mutated in Rothmund-Thomson syndrome, functions in telomere maintenance.

Telomeres are structures at the ends of chromosomes and are composed of long tracks of short tandem repeat DNA sequences bound by a unique set of proteins (shelterin). Telomeric DNA is believed to form G-quadruplex and D-loop structures, which presents a challenge to the DNA replication and repair machinery. Although the RecQ helicases WRN and BLM are implicated in the resolution of telomeric secondary structures, very little is known about RECQL4, the RecQ helicase mutated in Rothmund-Thomson syndrome (RTS). Here, we report that RTS patient cells have elevated levels of fragile telomeric ends and that RECQL4-depleted human cells accumulate fragile sites, sister chromosome exchanges, and double strand breaks at telomeric sites. Further, RECQL4 localizes to telomeres and associates with shelterin proteins TRF1 and TRF2. Using recombinant proteins we showed that RECQL4 resolves telomeric D-loop structures with the help of shelterin proteins TRF1, TRF2, and POT1. We also found a novel functional synergistic interaction of this protein with WRN during D-loop unwinding. These data implicate RECQL4 in telomere maintenance.

RecQ helicases in DNA double strand break repair and telomere maintenance.

Organisms are constantly exposed to various environmental insults which could adversely affect the stability of their genome. To protect their genomes against the harmful effect of these environmental insults, organisms have evolved highly diverse and efficient repair mechanisms. Defective DNA repair processes can lead to various kinds of chromosomal and developmental abnormalities. RecQ helicases are a family of evolutionarily conserved, DNA unwinding proteins which are actively engaged in various DNA metabolic processes, telomere maintenance and genome stability. Bacteria and lower eukaryotes, like yeast, have only one RecQ homolog, whereas higher eukaryotes including humans possess multiple RecQ helicases. These multiple RecQ helicases have redundant and/or non-redundant functions depending on the types of DNA damage and DNA repair pathways. Humans have five different RecQ helicases and defects in three of them cause autosomal recessive diseases leading to various kinds of cancer predisposition and/or aging phenotypes. Emerging evidence also suggests that the RecQ helicases have important roles in telomere maintenance. This review mainly focuses on recent knowledge about the roles of RecQ helicases in DNA double strand break repair and telomere maintenance which are important in preserving genome integrity.

Nucleotide excision repair of a DNA interstrand cross-link produces single- and double-strand breaks.

The DNA radical resulting from formal abstraction of a hydrogen atom from the thymidine methyl group, 5-(2'-deoxyuridinyl)methyl radical, forms interstrand cross-links with the opposing 2'-deoxyadenosine. This is the first chemically characterized, radical-mediated cross-link between two opposing nucleotides. In addition, cross-linking between opposing bases in the duplex is less common than between those separated by one or two nucleotides. The first step in cross-link repair was investigated using the UvrABC bacterial nucleotide excision repair system. UvrABC incised both strands of the cross-linked DNA, although the strand containing the cross-linked purine was preferred by the enzyme in two different duplexes. The incision sites in one strand were spaced 11-14 nucleotides apart, as is typical for UvrABC incision. The majority of incisions occur at the third phosphate from the 3'-side of the cross-link and eighth or ninth phosphate on the 5'-side. In addition, cleavage was found to occur on both strands, producing double-strand breaks in approximately 25-29% of the incision events. This is the first example of double-strand cleavage during nucleotide excision repair of cross-linked DNA that does not already contain a strand break in the vicinity of the cross-link.

The RECQL4 protein, deficient in Rothmund-Thomson syndrome is active on telomeric D-loops containing DNA metabolism blocking lesions.

Telomeres are critical for cell survival and functional integrity. Oxidative DNA damage induces telomeric instability and cellular senescence that are associated with normal aging and segmental premature aging disorders such as Werner Syndrome and Rothmund-Thomson Syndrome, caused by mutations in WRN and RECQL4 helicases respectively. Characterizing the metabolic roles of RECQL4 and WRN in telomere maintenance is crucial in understanding the pathogenesis of their associated disorders. We have previously shown that WRN and RECQL4 display a preference in vitro to unwind telomeric DNA substrates containing the oxidative lesion 8-oxoguanine. Here, we show that RECQL4 helicase has a preferential activity in vitro on telomeric substrates containing thymine glycol, a critical lesion that blocks DNA metabolism, and can be modestly stimulated further on a D-loop structure by TRF2, a telomeric shelterin protein. Unlike that reported for telomeric D-loops containing 8-oxoguanine, RECQL4 does not cooperate with WRN to unwind telomeric D-loops with thymine glycol, suggesting RECQL4 helicase is selective for the type of oxidative lesion. RECQL4's function at the telomere is not yet understood, and our findings suggest a novel role for RECQL4 in the repair of thymine glycol lesions to promote efficient telomeric maintenance.

Roles of Werner syndrome protein in protection of genome integrity.

Werner syndrome protein (WRN) is one of a family of five human RecQ helicases implicated in the maintenance of genome stability. The conserved RecQ family also includes RecQ1, Bloom syndrome protein (BLM), RecQ4, and RecQ5 in humans, as well as Sgs1 in Saccharomyces cerevisiae, Rqh1 in Schizosaccharomyces pombe, and homologs in Caenorhabditis elegans, Xenopus laevis, and Drosophila melanogaster. Defects in three of the RecQ helicases, RecQ4, BLM, and WRN, cause human pathologies linked with cancer predisposition and premature aging. Mutations in the WRN gene are the causative factor of Werner syndrome (WS). WRN is one of the best characterized of the RecQ helicases and is known to have roles in DNA replication and repair, transcription, and telomere maintenance. Studies both in vitro and in vivo indicate that the roles of WRN in a variety of DNA processes are mediated by post-translational modifications, as well as several important protein-protein interactions. In this work, we will summarize some of the early studies on the cellular roles of WRN and highlight the recent findings that shed some light on the link between the protein with its cellular functions and the disease pathology.

Conserved helicase domain of human RecQ4 is required for strand annealing-independent DNA unwinding.

Humans have five members of the well conserved RecQ helicase family: RecQ1, Bloom syndrome protein (BLM), Werner syndrome protein (WRN), RecQ4, and RecQ5, which are all known for their roles in maintaining genome stability. BLM, WRN, and RecQ4 are associated with premature aging and cancer predisposition. Of the three, RecQ4's biological and cellular roles have been least thoroughly characterized. Here we tested the helicase activity of purified human RecQ4 on various substrates. Consistent with recent results, we detected ATP-dependent RecQ4 unwinding of forked duplexes. However, our results provide the first evidence that human RecQ4's unwinding is independent of strand annealing, and that it does not require the presence of excess ssDNA. Moreover, we demonstrate that a point mutation of the conserved lysine in the Walker A motif abolished helicase activity, implying that not the N-terminal portion, but the helicase domain is solely responsible for the enzyme's unwinding activity. In addition, we demonstrate a novel stimulation of RecQ4's helicase activity by replication protein A, similar to that of RecQ1, BLM, WRN, and RecQ5. Together, these data indicate that specific biochemical activities and protein partners of RecQ4 are conserved with those of the other RecQ helicases.

Role of the guanine N1 imino proton in the migration and reaction of radical cations in DNA oligomers.

Oxidation of a guanine nucleobase to its radical cation in DNA oligomers causes an increase in the acidity of the N1 imino proton that may lead to its spontaneous transfer to N3 of the paired cytosine. This proton transfer is suspected of playing an important role in long-distance radical cation hopping in DNA and the decisive product-determining role in the reaction of the radical cation with H2O or O2. We prepared and investigated DNA oligomers in which certain deoxycytidines are replaced by 5-fluoro-2'-deoxycytidines (F5dC). The pKa of F5C was determined to be 1.7 units below that of dC, which causes proton transfer from the guanine radical cation to be thermodynamically unfavorable. Photoinitiated one-electron oxidation of the DNA by UV irradiation of a covalently attached anthraquinone derivative introduces a radical cation that hops throughout the oligomer and is trapped selectively at GG steps. The introduction of F5dC does not affect the efficiency of charge hopping, but it significantly reduces the amount of reaction at the GG sites, as revealed by subsequent reaction with formamidopyrimidine glycosylase. These findings suggest that transfer of the guanine radical cation N1 proton to cytosine does not play a significant role in long-range charge transfer, but this process does influence the reactions with H2O and/or O2.

One-electron oxidation of DNA oligomers that lack guanine: reaction and strand cleavage at remote thymines by long-distance radical cation hopping.

The anthraquinone (AQ) photosensitized one-electron oxidation of DNA introduces a radical cation (electron "hole") that migrates through the duplex by hopping. The radical cation normally is trapped irreversibly by reaction at guanine. We constructed AQ-linked DNA oligomers composed exclusively of A/T base pairs. Their irradiation led to reaction and strand cleavage primarily at thymines. Long-distance radical cation hopping to distant thymines was demonstrated by the distance dependence of the process and by experiments with DNA oligomers that contain a single remote GG step. The reaction of the radical cation at thymine was shown to involve its 5-methyl group by the replacement of selected thymines with uracils. These findings show that the reactivity of radical cations in DNA cannot be explained simply by exclusive reliance on the relative oxidation potential of the nucleobases. Instead, the site of reaction is determined in accord with the Curtin-Hammett principle for reactive species in rapid equilibrium.