Enzymes involved in DNA ligation and end-healing in the radioresistant bacterium Deinococcus radiodurans

BACKGROUND: Enzymes involved in DNA metabolic events of the highly radioresistant bacterium Deinococcus radiodurans are currently examined to understand the mechanisms that protect and repair the Deinococcus radiodurans genome after extremely high doses of gamma-irradiation. Although several Deinococcus radiodurans DNA repair enzymes have been characterised, no biochemical data is available for DNA ligation and DNA endhealing enzymes of Deinococcus radiodurans so far. DNA ligases are necessary to seal broken DNA backbones during replication, repair and recombination. In addition, ionizing radiation frequently leaves DNA strand-breaks that are not feasible for ligation and thus require end-healing by a 5'-polynucleotide kinase or a 3'-phosphatase. We expect that DNA ligases and end-processing enzymes play an important role in Deinococcus radiodurans DNA strand-break repair. RESULTS: In this report, we describe the cloning and expression of a Deinococcus radiodurans DNA ligase in Escherichia coli. This enzyme efficiently catalyses DNA ligation in the presence of Mn(II) and NAD+ as cofactors and lysine 128 was found to be essential for its activity. We have also analysed a predicted second DNA ligase from Deinococcus radiodurans that is part of a putative DNA repair operon and shows sequence similarity to known ATP-dependent DNA ligases. We show that this enzyme possesses an adenylyltransferase activity using ATP, but is not functional as a DNA ligase by itself. Furthermore, we identified a 5'-polynucleotide kinase similar to human polynucleotide kinase that probably prepares DNA termini for subsequent ligation. CONCLUSION: Deinococcus radiodurans contains a standard bacterial DNA ligase that uses NAD+ as a cofactor. Its enzymatic properties are similar to E. coli DNA ligase except for its preference for Mn(II) as a metal cofactor. The function of a putative second DNA ligase remains unclear, but its adenylyltransferase activity classifies it as a member of the nucleotidyltransferase family. Characterization of another protein from the same operon revealed a 5'-polynucleotide kinase with a possible role in DNA strand-break repair

Phosphorylation of human DNA polymerase λ by the cyclin-dependent kinase Cdk2/cyclin A complex is modulated by its association with proliferating cell nuclear antigen

DNA polymerase (Pol) λ is a member of the Pol X family and possesses four different enzymatic activities, being DNA polymerase, terminal transferase, deoxyribose phosphate lyase and polynucleotide synthetase, all localized in its C-terminal region. On the basis of its biochemical properties, Pol λ has been implicated in various DNA repair pathways, such as abasic site translesion DNA synthesis, base excision repair and non-homologous end joining of double strand breaks. However, its role in vivo has not yet been elucidated. In addition, Pol λ has been shown to interact with the replication clamp proliferating cell nuclear antigen (PCNA) in vitro and in vivo. In this work, we searched by affinity chromatography for novel partners and we identified the cyclin-dependent kinase Cdk2 as novel partner of Pol λ. Pol λ is phosphorylated in vitro by several Cdk/cyclin complexes, including Cdk2/cyclin A, in its proline-serine-rich domain. While the polymerase activity of Pol λ was not affected by Cdk2/cyclin A phosphorylation, phosphorylation of Pol λ was decreased by its interaction with PCNA. Finally, Pol λ is also phosphorylated in vivo in human cells and this phosphorylation is modulated during the cell cycl

Potassium layered polytitanates influence on low-pressure polyethylene properties in wide concentrations range

Thermoplastic composites based on low-pressure polyethylene and layered potassium titanates have been made in the work. We have shown the possibility to processing the composites into the products by injection molding at polytitanates content up to 50%wt. Technological properties of composites at low concentrations of filler are established. Layered potassium polytitanates influence on the mechanical and thermophysical properties of low-pressure polyethylene in a wide range of concentrations is investigated: from 1 to 50 %wt. Essential increase of complex of mechanical properties of polyethylene at introduction of small amounts of layered potassium polytitanates is shown. Due to the layered structure of the potassium polytitanates with particle size of about 500 nm in diameter and a thickness of less than 10 nm durability to shock loads increase comparable to the results of the introduction of fibrous potassiumhexatitanates is reached

Potassium layered polytitanates influence on low-pressure polyethylene properties in wide concentrations range

Thermoplastic composites based on low-pressure polyethylene and layered potassium titanates have been made in the work. We have shown the possibility to processing the composites into the products by injection molding at polytitanates content up to 50%wt. Technological properties of composites at low concentrations of filler are established. Layered potassium polytitanates influence on the mechanical and thermophysical properties of low-pressure polyethylene in a wide range of concentrations is investigated: from 1 to 50 %wt. Essential increase of complex of mechanical properties of polyethylene at introduction of small amounts of layered potassium polytitanates is shown. Due to the layered structure of the potassium polytitanates with particle size of about 500 nm in diameter and a thickness of less than 10 nm durability to shock loads increase comparable to the results of the introduction of fibrous potassiumhexatitanates is reached

Human replication protein A can suppress the intrinsic in vitro mutator phenotype of human DNA polymerase λ

DNA polymerase λ (pol λ) is a member of the X family DNA polymerases and is endowed with multiple enzymatic activities. In this work we investigated the in vitro miscoding properties of full-length, human pol λ either in the absence or in the presence of the human auxiliary proteins proliferating cell nuclear antigen (PCNA) and replication protein A (RP-A). Our data suggested that (i) pol λ had an intrinsic ability to create mismatches and to incorporate ribonucleotides at nearly physiological Mn(++) and Mg(++) concentrations; (ii) the sequence of the template-primer could influence the misincorporation frequency of pol λ; (iii) pol λ preferentially generated G:T and G:G mismatches; (iv) RP-A, but not PCNA, selectively prevented misincorporation of an incorrect nucleotide by pol λ, without affecting correct incorporation and (v) this inhibitory effect required a precise ratio between the concentrations of pol λ and RP-A. Possible physiological implications of these findings for the in vivo fidelity of pol λ are discussed

Physical and functional interactions between Werner syndrome helicase and mismatch-repair initiation factors

Werner syndrome (WS) is a severe recessive disorder characterized by premature aging, cancer predisposition and genomic instability. The gene mutated in WS encodes a bi-functional enzyme called WRN that acts as a RecQ-type DNA helicase and a 3′-5′ exonuclease, but its exact role in DNA metabolism is poorly understood. Here we show that WRN physically interacts with the MSH2/MSH6 (MutSα), MSH2/MSH3 (MutSβ) and MLH1/PMS2 (MutLα) heterodimers that are involved in the initiation of mismatch repair (MMR) and the rejection of homeologous recombination. MutSα and MutSβ can strongly stimulate the helicase activity of WRN specifically on forked DNA structures with a 3′-single-stranded arm. The stimulatory effect of MutSα on WRN-mediated unwinding is enhanced by a G/T mismatch in the DNA duplex ahead of the fork. The MutLα protein known to bind to the MutS α-heteroduplex complexes has no effect on WRN-mediated DNA unwinding stimulated by MutSα, nor does it affect DNA unwinding by WRN alone. Our data are consistent with results of genetic experiments in yeast suggesting that MMR factors act in conjunction with a RecQ-type helicase to reject recombination between divergent sequence

Passive microwave radiometry in biomedical studies

Passive microwave radiometry (MWR) measures natural emissions in the range 1–10 GHz from proteins, cells, organs and the whole human body. The intensity of intrinsic emission is determined by biochemical and biophysical processes. The nature of this process is still not very well known. Infrared thermography (IRT) can detect emission several microns deep (skin temperature), whereas MWR allows detection of thermal abnormalities down to several centimeters (internal or deep temperature). MWR is noninvasive and inexpensive. It requires neither fluorescent nor radioactive labels, nor ionizing or other radiation. MWR can be used in early drug discovery as well as preclinical and clinical studies

MRE11 complex links RECQ5 helicase to sites of DNA damage

RECQ5 DNA helicase suppresses homologous recombination (HR) possibly through disruption of RAD51 filaments. Here, we show that RECQ5 is constitutively associated with the MRE11-RAD50-NBS1 (MRN) complex, a primary sensor of DNA double-strand breaks (DSBs) that promotes DSB repair and regulates DNA damage signaling via activation of the ATM kinase. Experiments with purified proteins indicated that RECQ5 interacts with the MRN complex through both MRE11 and NBS1. Functional assays revealed that RECQ5 specifically inhibited the 3′→5′ exonuclease activity of MRE11, while MRN had no effect on the helicase activity of RECQ5. At the cellular level, we observed that the MRN complex was required for the recruitment of RECQ5 to sites of DNA damage. Accumulation of RECQ5 at DSBs was neither dependent on MDC1 that mediates binding of MRN to DSB-flanking chromatin nor on CtIP that acts in conjunction with MRN to promote resection of DSBs for repair by HR. Collectively, these data suggest that the MRN complex recruits RECQ5 to sites of DNA damage to regulate DNA repai

RECQ5 helicase associates with the C-terminal repeat domain of RNA polymerase II during productive elongation phase of transcription

It is known that transcription can induce DNA recombination, thus compromising genomic stability. RECQ5 DNA helicase promotes genomic stability by regulating homologous recombination. Recent studies have shown that RECQ5 forms a stable complex with RNA polymerase II (RNAPII) in human cells, but the cellular role of this association is not understood. Here, we provide evidence that RECQ5 specifically binds to the Ser2,5-phosphorylated C-terminal repeat domain (CTD) of the largest subunit of RNAPII, RPB1, by means of a Set2-Rpb1-interacting (SRI) motif located at the C-terminus of RECQ5. We also show that RECQ5 associates with RNAPII-transcribed genes in a manner dependent on the SRI motif. Notably, RECQ5 density on transcribed genes correlates with the density of Ser2-CTD phosphorylation, which is associated with the productive elongation phase of transcription. Furthermore, we show that RECQ5 negatively affects cell viability upon inhibition of spliceosome assembly, which can lead to the formation of mutagenic R-loop structures. These data indicate that RECQ5 binds to the elongating RNAPII complex and support the idea that RECQ5 plays a role in the maintenance of genomic stability during transcriptio

RECQ5 helicase associates with the C-terminal repeat domain of RNA polymerase II during productive elongation phase of transcription

It is known that transcription can induce DNA recombination, thus compromising genomic stability. RECQ5 DNA helicase promotes genomic stability by regulating homologous recombination. Recent studies have shown that RECQ5 forms a stable complex with RNA polymerase II (RNAPII) in human cells, but the cellular role of this association is not understood. Here, we provide evidence that RECQ5 specifically binds to the Ser2,5-phosphorylated C-terminal repeat domain (CTD) of the largest subunit of RNAPII, RPB1, by means of a Set2–Rpb1-interacting (SRI) motif located at the C-terminus of RECQ5. We also show that RECQ5 associates with RNAPII-transcribed genes in a manner dependent on the SRI motif. Notably, RECQ5 density on transcribed genes correlates with the density of Ser2-CTD phosphorylation, which is associated with the productive elongation phase of transcription. Furthermore, we show that RECQ5 negatively affects cell viability upon inhibition of spliceosome assembly, which can lead to the formation of mutagenic R-loop structures. These data indicate that RECQ5 binds to the elongating RNAPII complex and support the idea that RECQ5 plays a role in the maintenance of genomic stability during transcription