scholarly journals CtIP promotes the motor activity of DNA2 to accelerate long-range DNA end resection

2020 ◽  
Vol 117 (16) ◽  
pp. 8859-8869 ◽  
Author(s):  
Ilaria Ceppi ◽  
Sean M. Howard ◽  
Kristina Kasaciunaite ◽  
Cosimo Pinto ◽  
Roopesh Anand ◽  
...  
Keyword(s):  
Motor Activity ◽  
Long Range ◽  
Single Molecule ◽  
Short Range ◽  
Atp Hydrolysis ◽  
Strand Break ◽  
Motor Activities ◽  
Dna Strands ◽  
Dna End Resection ◽  
Wrn Helicase

To repair a DNA double-strand break by homologous recombination, 5′-terminated DNA strands must first be resected to reveal 3′-overhangs. This process is initiated by a short-range resection catalyzed by MRE11-RAD50-NBS1 (MRN) stimulated by CtIP, which is followed by a long-range step involving EXO1 or DNA2 nuclease. DNA2 is a bifunctional enzyme that contains both single-stranded DNA (ssDNA)-specific nuclease and motor activities. Upon DNA unwinding by Bloom (BLM) or Werner (WRN) helicase, RPA directs the DNA2 nuclease to degrade the 5′-strand. RPA bound to ssDNA also represents a barrier, explaining the need for the motor activity of DNA2 to displace RPA prior to resection. Using ensemble and single-molecule biochemistry, we show that CtIP also dramatically stimulates the adenosine 5′-triphosphate (ATP) hydrolysis-driven motor activity of DNA2 involved in the long-range resection step. This activation in turn strongly promotes the degradation of RPA-coated ssDNA by DNA2. Accordingly, the stimulatory effect of CtIP is only observed with wild-type DNA2, but not the helicase-deficient variant. Similarly to the function of CtIP to promote MRN, also the DNA2 stimulatory effect is facilitated by CtIP phosphorylation. The domain of CtIP required to promote DNA2 is located in the central region lacking in lower eukaryotes and is fully separable from domains involved in the stimulation of MRN. These results establish how CtIP couples both MRE11-dependent short-range and DNA2-dependent long-range resection and define the involvement of the motor activity of DNA2 in this process. Our data might help explain the less severe resection defects of MRE11 nuclease-deficient cells compared to those lacking CtIP.

scholarly journals CtIP promotes the motor activity of DNA2 to accelerate long-range DNA end resection

2019 ◽  
Author(s):  
Ilaria Ceppi ◽  
Sean M. Howard ◽  
Kristina Kasaciunaite ◽  
Cosimo Pinto ◽  
Roopesh Anand ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Motor Activity ◽  
Long Range ◽  
Single Molecule ◽  
Atp Hydrolysis ◽  
Dna Unwinding ◽  
List Type ◽  
Mrn Complex ◽  
Dna End Resection ◽  
End Resection

SummaryBLM or WRN helicases function with the DNA2 helicase-nuclease to resect DNA doublestrand breaks and initiate homologous recombination. Upon DNA unwinding by BLM/WRN, RPA directs the DNA2 nuclease to degrade the 5’-strand, revealing the 3’ overhang needed for recombination. RPA bound to ssDNA also represents a barrier, explaining the need for the motor activity of DNA2 to displace RPA prior to resection. Using ensemble and single molecule biochemistry, we show that phosphorylated CtIP dramatically stimulates the ATP hydrolysis driven motor activity of DNA2. This activation in turn strongly promotes the degradation of RPA-coated ssDNA by DNA2. The domains of CtIP required to stimulate DNA2 are separable from those that regulate the MRN complex. These results establish that CtIP couples both MRE11-dependent short and DNA2-dependent long-range resection, and show how the motor activity of DNA2 promotes resection. Our data explain the less severe resection defects of MRE11 nuclease-deficient cells compared to those lacking CtIP.HighlightsPhosphorylated CtIP stimulates the motor activity of DNA2The activated DNA2 translocase facilitates degradation of RPA-coated ssDNACtIP promotes both MRN and DNA2 nucleases coupling short and long-range resectionThe CtIP domains required to promote DNA2 and MRN are distinct and fully separable

scholarly journals Structures and single-molecule analysis of bacterial motor nuclease AdnAB illuminate the mechanism of DNA double-strand break resection

2019 ◽  
Vol 116 (49) ◽  
pp. 24507-24516 ◽  
Author(s):  
Ning Jia ◽  
Mihaela C. Unciuleac ◽  
Chaoyou Xue ◽  
Eric C. Greene ◽  
Dinshaw J. Patel ◽  
...  
Keyword(s):  
Single Molecule ◽  
Atp Hydrolysis ◽  
Strand Break ◽  
Dna Double Strand Breaks ◽  
Iron Sulfur Cluster ◽  
Strand Breaks ◽  
Nuclease Domain ◽  
Dna Double Strand Break ◽  
Before And After ◽  
Single Molecule Analysis

Mycobacterial AdnAB is a heterodimeric helicase–nuclease that initiates homologous recombination by resecting DNA double-strand breaks (DSBs). The AdnA and AdnB subunits are each composed of an N-terminal motor domain and a C-terminal nuclease domain. Here we report cryoelectron microscopy (cryo-EM) structures of AdnAB in three functional states: in the absence of DNA and in complex with forked duplex DNAs before and after cleavage of the 5′ single-strand DNA (ssDNA) tail by the AdnA nuclease. The structures reveal the path of the 5′ ssDNA through the AdnA nuclease domain and the mechanism of 5′ strand cleavage; the path of the 3′ tracking strand through the AdnB motor and the DNA contacts that couple ATP hydrolysis to mechanical work; the position of the AdnA iron–sulfur cluster subdomain at the Y junction and its likely role in maintaining the split trajectories of the unwound 5′ and 3′ strands. Single-molecule DNA curtain analysis of DSB resection reveals that AdnAB is highly processive but prone to spontaneous pausing at random sites on duplex DNA. A striking property of AdnAB is that the velocity of DSB resection slows after the enzyme experiences a spontaneous pause. Our results highlight shared as well as distinctive properties of AdnAB vis-à-vis the RecBCD and AddAB clades of bacterial DSB-resecting motor nucleases.

scholarly journals The chromatin remodeler Chd1 supports MRX and Exo1 functions in resection of DNA double-strand breaks

PLoS Genetics ◽  
2021 ◽  
Vol 17 (9) ◽  
pp. e1009807
Author(s):  
Marco Gnugnoli ◽  
Erika Casari ◽  
Maria Pia Longhese
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Atpase Activity ◽  
Long Range ◽  
Chromatin Remodeling ◽  
Short Range ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Dsb Repair ◽  
Strand Breaks ◽  
Dna Strands

Repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) requires that the 5’-terminated DNA strands are resected to generate single-stranded DNA overhangs. This process is initiated by a short-range resection catalyzed by the MRX (Mre11-Rad50-Xrs2) complex, which is followed by a long-range step involving the nucleases Exo1 and Dna2. Here we show that the Saccharomyces cerevisiae ATP-dependent chromatin-remodeling protein Chd1 participates in both short- and long-range resection by promoting MRX and Exo1 association with the DSB ends. Furthermore, Chd1 reduces histone occupancy near the DSB ends and promotes DSB repair by HR. All these functions require Chd1 ATPase activity, supporting a role for Chd1 in the opening of chromatin at the DSB site to facilitate MRX and Exo1 processing activities.

scholarly journals Competing interactions modulate the activity of Sgs1 during DNA end resection

2019 ◽  
Author(s):  
Kristina Kasaciunaite ◽  
Fergus Fettes ◽  
Maryna Levikova ◽  
Peter Daldrop ◽  
Petr Cejka ◽  
...  
Keyword(s):  
Single Molecule ◽  
Genome Stability ◽  
Strand Break ◽  
Cellular Functions ◽  
Single Stranded Dna ◽  
Single Molecule Level ◽  
Protein Partners ◽  
Dna Double Strand Break ◽  
Dna End Resection ◽  
End Resection

AbstractDNA double-strand break repair by homologous recombination employs long-range resection of the 5’ DNA ends at the break points. In Saccharomyces cerevisiae, this process can be performed by the RecQ helicase Sgs1 and the helicase-nuclease Dna2. Though functional interplay has been shown, it remains unclear whether and how the proteins cooperate on the molecular level. Here, we resolved the dynamics of DNA unwinding by Sgs1 at the single molecule level and investigated its regulation by Dna2, the single-stranded DNA binding protein RPA and the Top3-Rmi1 complex. We found that Dna2 modulates the velocity of Sgs1, indicating that during end resection the proteins form a physical complex and couple their activities. Sgs1 unwinds DNA and feeds single-stranded DNA to Dna2 for degradation. RPA is found to regulate the processivity and the affinity of Sgs1 to the DNA fork, while Top3-Rmi1 modulated the velocity of Sgs1. We think that the differential regulation of the Sgs1 activity by its protein partners is important to allow diverse cellular functions of Sgs1 during the maintenance of genome stability.

scholarly journals Polymerase theta-helicase promotes end joining by stripping single-stranded DNA-binding proteins and bridging DNA ends

2021 ◽  
Author(s):  
Jeffrey M Schaub ◽  
Michael M Soniat ◽  
Ilya J Finkelstein
Keyword(s):  
Single Molecule ◽  
Protein A ◽  
Strand Break ◽  
Helicase Domain ◽  
End Joining ◽  
Single Stranded Dna ◽  
Double Stranded Dna ◽  
Dna Strands ◽  
In Trans

Homologous recombination-deficient cancers rely on DNA polymerase Theta (Polθ)-Mediated End Joining (TMEJ), an alternative double-strand break repair pathway. Polθ is the only vertebrate polymerase that encodes an N-terminal superfamily 2 (SF2) helicase domain, but the role of this helicase domain in TMEJ remains unclear. Using single-molecule imaging, we demonstrate that Polθ-helicase (Polθ-h) is a highly processive single-stranded DNA (ssDNA) motor protein that can efficiently strip Replication Protein A (RPA) from ssDNA. Polθ-h also has a limited capacity for disassembling RAD51 filaments but is not processive on double- stranded DNA. Polθ-h can bridge two non-complementary DNA strands in trans. PARylation of Polθ-h by PARP-1 resolves these DNA bridges. We conclude that Polθ-h removes RPA and RAD51 filaments and mediates bridging of DNA overhangs to aid in polymerization by the Polθ polymerase domain.

scholarly journals A DNA nick at Ku-blocked double-strand break ends serves as an entry site for exonuclease 1 (Exo1) or Sgs1–Dna2 in long-range DNA end resection

2018 ◽  
Vol 293 (44) ◽  
pp. 17061-17069 ◽  
Author(s):  
Weibin Wang ◽  
James M. Daley ◽  
Youngho Kwon ◽  
Xiaoyu Xue ◽  
Danielle S. Krasner ◽  
...  
Keyword(s):  
Long Range ◽  
Current Model ◽  
Specific Interaction ◽  
Protein A ◽  
Nuclease Activity ◽  
Strand Break ◽  
Entry Site ◽  
Exonuclease 1 ◽  
Dna End Resection ◽  
End Resection

The repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) is initiated by nucleolytic resection of the DNA break ends. The current model, being based primarily on genetic analyses in Saccharomyces cerevisiae and companion biochemical reconstitution studies, posits that end resection proceeds in two distinct stages. Specifically, the initiation of resection is mediated by the nuclease activity of the Mre11–Rad50–Xrs2 (MRX) complex in conjunction with its cofactor Sae2, and long-range resection is carried out by exonuclease 1 (Exo1) or the Sgs1–Top3–Rmi1–Dna2 ensemble. Using fully reconstituted systems, we show here that DNA with ends occluded by the DNA end-joining factor Ku70–Ku80 becomes a suitable substrate for long-range 5′–3′ resection when a nick is introduced at a locale proximal to one of the Ku-bound DNA ends. We also show that Sgs1 can unwind duplex DNA harboring a nick, in a manner dependent on a species-specific interaction with the ssDNA-binding factor replication protein A (RPA). These biochemical systems and results will be valuable for guiding future endeavors directed at delineating the mechanistic intricacy of DNA end resection in eukaryotes.

Structural dynamics of P-type ATPase ion pumps

2019 ◽  
Vol 47 (5) ◽  
pp. 1247-1257 ◽  
Author(s):  
Mateusz Dyla ◽  
Sara Basse Hansen ◽  
Poul Nissen ◽  
Magnus Kjaergaard
Keyword(s):  
Structural Dynamics ◽  
Single Molecule ◽  
New Technologies ◽  
Atp Hydrolysis ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Time Resolved ◽  
Dynamics Simulations ◽  
Types Of Information ◽  
P Type

Abstract P-type ATPases transport ions across biological membranes against concentration gradients and are essential for all cells. They use the energy from ATP hydrolysis to propel large intramolecular movements, which drive vectorial transport of ions. Tight coordination of the motions of the pump is required to couple the two spatially distant processes of ion binding and ATP hydrolysis. Here, we review our current understanding of the structural dynamics of P-type ATPases, focusing primarily on Ca2+ pumps. We integrate different types of information that report on structural dynamics, primarily time-resolved fluorescence experiments including single-molecule Förster resonance energy transfer and molecular dynamics simulations, and interpret them in the framework provided by the numerous crystal structures of sarco/endoplasmic reticulum Ca2+-ATPase. We discuss the challenges in characterizing the dynamics of membrane pumps, and the likely impact of new technologies on the field.

scholarly journals Structural basis of long-range to short-range synaptic transition in NHEJ

Nature ◽  
2021 ◽  
Author(s):  
Siyu Chen ◽  
Linda Lee ◽  
Tasmin Naila ◽  
Susan Fishbain ◽  
Annie Wang ◽  
...  
Keyword(s):  
Long Range ◽  
Short Range ◽  
Structural Basis

scholarly journals Comparison of short-range order in irradiated dysprosium titanates

2021 ◽  
Vol 5 (1) ◽  
Author(s):  
Roman Sherrod ◽  
Eric C. O’Quinn ◽  
Igor M. Gussev ◽  
Cale Overstreet ◽  
Joerg Neuefeind ◽  
...  
Keyword(s):  
Long Range ◽  
Short Range ◽  
Structural Model ◽  
Ion Irradiation ◽  
Function Analysis ◽  
Ion Beam ◽  
Structural Response ◽  
Heavy Ion ◽  
Range Order ◽  
Radiation Resistant

AbstractThe structural response of Dy2TiO5 oxide under swift heavy ion irradiation (2.2 GeV Au ions) was studied over a range of structural length scales utilizing neutron total scattering experiments. Refinement of diffraction data confirms that the long-range orthorhombic structure is susceptible to ion beam-induced amorphization with limited crystalline fraction remaining after irradiation to 8 × 1012 ions/cm2. In contrast, the local atomic arrangement, examined through pair distribution function analysis, shows only subtle changes after irradiation and is still described best by the original orthorhombic structural model. A comparison to Dy2Ti2O7 pyrochlore oxide under the same irradiation conditions reveals a different behavior: while the dysprosium titanate pyrochlore is more radiation resistant over the long-range with smaller degree of amorphization as compared to Dy2TiO5, the former involves more local atomic rearrangements, best described by a pyrochlore-to-weberite-type transformation. These results highlight the importance of short-range and medium-range order analysis for a comprehensive description of radiation behavior.

SHORT-RANGE AND LONG-RANGE ORDER OF TITANIUM IN Ti1+xS2

1977 ◽  
Vol 38 (C7) ◽  
pp. C7-202-C7-206 ◽  
Author(s):  
R. MORET ◽  
M. HUBER ◽  
R. COMÈS
Keyword(s):  
Long Range ◽  
Short Range ◽  
Range Order ◽  
Long Range Order
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