The three-dimensional structure of the HRDC domain and implications for the Werner and Bloom syndrome proteins

RecQ helicases are key genome maintenance enzymes that function in DNA replication, recombination, and repair. In contrast to nearly every other identified RecQ family member, the RecQ helicase from the radioresistant bacterium Deinococcus radiodurans encodes three "Helicase and RNase D C-terminal" (HRDC) domains at its C terminus. HRDC domains have been implicated in structure-specific nucleic acid binding with roles in targeting RecQ proteins to particular DNA structures; however, only RecQ proteins with single HRDC domains have been examined to date. We demonstrate that the HRDC domains can be proteolytically removed from the D. radiodurans RecQ (DrRecQ) C terminus, consistent with each forming a structural domain. Using this observation as a guide, we produced a panel of recombinant DrRecQ variants lacking combinations of its HRDC domains to investigate their biochemical functions. The N-terminal-most HRDC domain is shown to be critical for high affinity DNA binding and for efficient unwinding of DNA in some contexts. In contrast, the more C-terminal HRDC domains attenuate the DNA binding affinity and DNAdependent ATP hydrolysis rate of the enzyme and play more complex roles in structure-specific DNA unwinding. Our results indicate that the multiple DrRecQ HRDC domains have evolved to encode DNA binding and regulatory functions in the enzyme.

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Comparison Of Ecrecq and Drrecq Hrdc Domains-mentioning

confidence: 99%

See 1 more Smart Citation

Three HRDC Domains Differentially Modulate Deinococcus radiodurans RecQ DNA Helicase Biochemical Activity

Killoran

Keck

2006

“…The HRDC (helicase-and-ribonuclease D/C-terminal) domain is found in a number of RecQ helicases and displays significant sequence variability. The three known structures of HRDC, from E. coli RecQ (54), yeast Sgs1 (55), and WRN (56), all reveal a helical bundle that, superficially at least, resembles that of UvsW.1. The role of the HRDC is currently ill-defined but one suggestion is that it provides substrate recognition elements to enhance that of the parent SF2 helicase.…”

Section: Discussionmentioning

confidence: 99%

Crystallographic and NMR Analyses of UvsW and UvsW.1 from Bacteriophage T4

Kerr

Sivakolundu

et al. 2007

The uvsWXY system is implicated in the replication and repair of the bacteriophage T4 genome. Whereas the roles of the recombinase (UvsX) and the recombination mediator protein (UvsY) are known, the precise role of UvsW is unclear. Sequence analysis identifies UvsW as a member of the monomeric SF2 helicase superfamily that translocates nucleic acid substrates via the action of two RecA-like motor domains. Functional homologies to Escherichia coli RecG and biochemical analyses have shown that UvsW interacts with branched nucleic acid substrates, suggesting roles in recombination and the rescue of stalled replication forks. A sequencing error at the 3-end of the uvsW gene has revealed a second, short open reading frame that encodes a protein of unknown function called UvsW.1. We have determined the crystal structure of UvsW to 2.7 Å and the NMR solution structure of UvsW.1. UvsW has a four-domain architecture with structural homology to the eukaryotic SF2 helicase, Rad54. A model of the UvsW-ssDNA complex identifies structural elements and conserved residues that may interact with nucleic acid substrates. The NMR solution structure of UvsW.1 reveals a dynamic four-helix bundle with homology to the structure-specific nucleic acid binding module of RecQ helicases.Bacteriophage T4 remains an ideal model system in which to study the fundamental mechanisms of nucleic acid metabolism. DNA replication is particularly well suited for study in T4because the viral genome encodes all the necessary replicative enzymes and accessory proteins that exist in higher organisms (1). During the early stage of the T4 infection cycle, DNA synthesis is initiated by a classic origin-dependent mechanism in which replication complexes are assembled onto persistent R-loops (RNA-DNA hybrids) (2). However, this mechanism is quickly abandoned, and during the later stages of infection the switch is made to the more efficient recombination-dependent replication (RDR) 6 to synthesize the bulk of the DNA (3). RDR produces a complex concatemeric network of T4 DNA that is ultimately resolved into the circularly permuted and terminally redundant genome that exists within the mature virus particles. The initiation stage of RDR is a recombination reaction in which the 3Ј-end of a single-stranded region of T4 DNA invades a homologous region of DNA to generate a displacement (D) loop intermediate. The T4 replication machinery is then assembled within the D loop to begin replication from the invading 3Ј-end (3).Homologous recombination is a key process in DNA metabolism that remains poorly understood at the molecular level, and T4 is an ideal system in which to study the mechanism due to its central role in RDR and T4 replication. The T4 proteins UvsX and UvsY perform equivalent functions to the recombination proteins Rad51 and Rad52, respectively, in eukaryotic systems (4). UvsX and UvsY are both members of the uvsWXY system, which is associated with susceptibility to DNA damage because of UV radiation (hence the name). This is consistent with the role...

“…Like RecQ family member Werner protein, BLM contains a conserved domain called RQC (RecQ C-terminal) that forms a winged-helix structure exploited to bind DNA (23). Additionally, BLM contains a motif called HRDC (helicase and RNase D C-terminal) that when folded creates another DNA binding site (24,25). The multiple DNA binding domains allow BLM to bind two or more single-stranded DNA oligomers simultaneously, bringing them together and facilitating strand annealing.…”

mentioning

confidence: 99%

Mechanisms by Which Bloom Protein Can Disrupt Recombination Intermediates of Okazaki Fragment Maturation

Bartos

Wang

Pike

et al. 2006

Bloom syndrome is a familial genetic disorder associated with sunlight sensitivity and a high predisposition to cancers. The mutated gene, Bloom protein (BLM), encodes a DNA helicase that functions in genome maintenance via roles in recombination repair and resolution of recombination structures. We designed substrates representing illegitimate recombination intermediates formed when a displaced DNA flap generated during maturation of Okazaki fragments escapes cleavage by flap endonuclease-1 and anneals to a complementary ectopic DNA site. Results show that displaced, replication protein A (RPA)-coated flaps could readily bind and ligate at the complementary site to initiate recombination. RPA also displayed a strand-annealing activity that hastens the rate of recombination intermediate formation. BLM helicase activity could directly disrupt annealing at the ectopic site and promote flap endonuclease-1 cleavage. Additionally, BLM has its own strand-annealing and strand-exchange activities. RPA inhibited the BLM strand-annealing activity, thereby promoting helicase activity and complex dissolution. BLM strand exchange could readily dissociate invading flaps, e.g. in a D-loop, if the exchange step did not involve annealing of RPA-coated strands. Use of ATP to activate the helicase function did not aid flap displacement by exchange, suggesting that this is a helicase-independent mechanism of complex dissociation. When RPA could bind, it displayed its own strand-exchange activity. We interpret these results to explain how BLM is well equipped to deal with alternative recombination intermediate structures. Bloom protein (BLM)2 is a member of the RecQ family of 3Ј-5Ј helicases that assist in maintaining genome stability. Mutation or loss of function of the BLM protein causes Bloom syndrome (BS), an autosomal recessive disease characterized by sunlight sensitivity, proportional dwarfism, and a high predisposition toward many different types of cancer (1). Cells with BLM deficiency show increased chromosomal abnormalities, including hyper-recombination, elevated rates of sister chromatid exchange, and the abnormal accumulation of replication intermediates, resulting in an increase in the overall level of genomic instability (2-4). Knock-out of BLM in mice causes embryonic lethality, whereas some mutations produce live mice prone to tumorigenesis (5, 6).BLM plays a role in several critical genome maintenance pathways. Immunodepletion of Xenopus BLM inhibits the replication of DNA in reconstituted nuclei, suggesting that BLM is directly involved in DNA replication (7). Telomere proteins TRF2 and TRF1 colocalize with BLM in immortalized cells lines and regulate its helicase activity in vivo, signifying a role for BLM in telomere maintenance (8). BLM assists in the recovery of stalled replication forks and in the prevention of repeat expansion by stabilizing repeated sequences (9 -13). Additionally, BLM has been proposed to promote proper intermediate resolution and suppress crossovers in the homologous recombination pathwa...