Molecular watchdogs on genome patrol

By removing various obstacles from single strands of DNA, an enzyme called Pif1 clears the way for other enzymes that act on DNA

An attempt to measure the time delays of three gravitational lenses

Thesis (S.B.)--Massachusetts Institute of Technology, Dept. of Physics, 2007.Includes bibliographical references (p. 95-97).I present the results of reduction and analysis of two seasons of gravitational lens monitoring using the Very Large Array (VLA) at 8.5 GHz. The campaign monitored five gravitational lenses, GL1608, GL1830, GL1632, GL1838, and GL2004 from 24 January 2002 until 18 September 2002, and from 21 May 2003 until 29 January 2004. In addition to gravitational lenses, the campaign monitored ten flux and phase calibrators. The goal of this work was to measure the gravitational lens time delays. The ultimate goal was to estimate H₀ in a one-step calculation as proposed by Refsdal in 1964 [30]. I reduced the data using AIPS and DIFMAP astronomical data processing software. I analyzed the final light curves in MATLAB using Pelt's non-interpolative dispersion method [33]. Monte Carlo simulations were used to verify the results. I focused my analysis on three lenses: GL1632, GL1838, and GL2004. Two gravitational lenses, GL1632, and GL1838 exhibited significant flux variability and I was able to measure tentative time delay for these lenses. My analysis suggests a time delay of ... days. I used this value and the lens model by Winn et al. [15] to calculate H₀=... for a flat cosmological model with ... For GL1838, I calculated a tentative time delay of ... days. Combined with Winn's lens model, this tentative measurement gives H₀... Unfortunately the GL1838 time delay calculation was based on a light curve feature at the end of Season 2 and is not very reliable. The flux density of GL2004 images varied very little over the course of the campaign and it was not possible to calculate its time delay. However, we observed an interesting pattern of variability in light curves suggesting that GL2004 is probably subject to differential Galactic scintillation. Our observations show that GL1838 and GL1632 experience significant flux density variations on timescales of months, so it should be possible to measure their time delay more accurately in future monitoring campaigns.by Gheorghe Chistol.S.B

TRAIP is a master regulator of DNA interstrand crosslink repair

Cells often use multiple pathways to repair the same DNA lesion, and the choice of pathway has substantial implications for the fidelity of genome maintenance. DNA interstrand crosslinks covalently link the two strands of DNA, and thereby block replication and transcription; the cytotoxicity of these crosslinks is exploited for chemotherapy. In Xenopus egg extracts, the collision of replication forks with interstrand crosslinks initiates two distinct repair pathways. NEIL3 glycosylase can cleave the crosslink; however, if this fails, Fanconi anaemia proteins incise the phosphodiester backbone that surrounds the interstrand crosslink, generating a double-strand-break intermediate that is repaired by homologous recombination. It is not known how the simpler NEIL3 pathway is prioritized over the Fanconi anaemia pathway, which can cause genomic rearrangements. Here we show that the E3 ubiquitin ligase TRAIP is required for both pathways. When two replisomes converge at an interstrand crosslink, TRAIP ubiquitylates the replicative DNA helicase CMG (the complex of CDC45, MCM2–7 and GINS). Short ubiquitin chains recruit NEIL3 through direct binding, whereas longer chains are required for the unloading of CMG by the p97 ATPase, which enables the Fanconi anaemia pathway. Thus, TRAIP controls the choice between the two known pathways of replication-coupled interstrand-crosslink repair. These results, together with our other recent findings establish TRAIP as a master regulator of CMG unloading and the response of the replisome to obstacles

TRAIP is a master regulator of DNA interstrand crosslink repair

Cells often use multiple pathways to repair the same DNA lesion, and the choice of pathway has substantial implications for the fidelity of genome maintenance. DNA interstrand crosslinks covalently link the two strands of DNA, and thereby block replication and transcription; the cytotoxicity of these crosslinks is exploited for chemotherapy. In Xenopus egg extracts, the collision of replication forks with interstrand crosslinks initiates two distinct repair pathways. NEIL3 glycosylase can cleave the crosslink; however, if this fails, Fanconi anaemia proteins incise the phosphodiester backbone that surrounds the interstrand crosslink, generating a double-strand-break intermediate that is repaired by homologous recombination. It is not known how the simpler NEIL3 pathway is prioritized over the Fanconi anaemia pathway, which can cause genomic rearrangements. Here we show that the E3 ubiquitin ligase TRAIP is required for both pathways. When two replisomes converge at an interstrand crosslink, TRAIP ubiquitylates the replicative DNA helicase CMG (the complex of CDC45, MCM2–7 and GINS). Short ubiquitin chains recruit NEIL3 through direct binding, whereas longer chains are required for the unloading of CMG by the p97 ATPase, which enables the Fanconi anaemia pathway. Thus, TRAIP controls the choice between the two known pathways of replication-coupled interstrand-crosslink repair. These results, together with our other recent findings establish TRAIP as a master regulator of CMG unloading and the response of the replisome to obstacles

Dissecting the Operating Mechanism of a Biological Motor One Molecule at a Time

Double-stranded DNA viruses, including most bacteriophages and mammalian herpesviruses, package their genomes into a pre-formed protein capsid during their self-assembly. DNA is compacted to near-crystalline densities at the end of packaging. This remarkable mechanical task is performed by a powerful ATP-driven molecular machine known as the packaging motor. Bacteriophage Phi29, a model system for studying DNA packaging, has a 19.3-kbp genome and its packaging motor is composed of a connector, an RNA scaffold, and a pentameric ring ATPase.Ring ATPases of the ASCE superfamily perform a variety of cellular functions. An important question about the operation of these molecular machines is how the ring subunits coordinate their chemical and mechanical transitions. Here we present the first comprehensive mechanochemical characterization of a homomeric ring ATPase - Phi29 gp16 - which translocates dsDNA in cycles composed of alternating dwells and bursts. We use high-resolution optical tweezers to determine the effect of nucleotide analogs on the cycle. We find that ATP hydrolysis occurs sequentially during the burst and that ADP release is interlaced with ATP binding during the dwell, revealing a high degree of coordination among ring subunits. Moreover, we show that the motor displays an unexpected division of labor: although all subunits of the homo-pentamer bind and hydrolyze ATP during each cycle, only four participate in translocation whereas the remaining subunit plays an ATP-dependent regulatory role.Several viral packaging motors have been shown to slow down as the capsid fills up with DNA, but it remains unclear how the packaging velocity is regulated. Here we use high-resolution optical tweezers to monitor the base-pair-scale packaging dynamics at various degrees of capsid filling. By comparing the burst duration at various degrees of capsid filling and different external forces, we estimate an internal force of ~20 pN at 100% filling, much lower than the motor's stall force. We find that the motor's step size is continuously modulated by capsid filling, in quantitative agreement with measurements of DNA rotation by the Phi29 packaging motor. In addition, we find the motor switches on and off at high filling by entering into long-lived pauses, which may allow DNA relaxation within the capsid. Together, our results reveal that the motor is not passively stalled by a large internal force at high filling as suggested by previous models. Instead, the motor is actively throttled down via several mechanisms in response to DNA encapsidation. The intricate crosstalk between the motor and the capsid plays a key role in orchestrating the molecular events leading to packaging termination and virus maturation, and may represent a general design principle shared by different viruses

Two-subunit DNA escort mechanism and inactive subunit bypass in an ultra-fast ring ATPase.

SpoIIIE is a homo-hexameric dsDNA translocase responsible for completing chromosome segregation in Bacillus subtilis. Here, we use a single-molecule approach to monitor SpoIIIE translocation when challenged with neutral-backbone DNA and non-hydrolyzable ATP analogs. We show that SpoIIIE makes multiple essential contacts with phosphates on the 5'→3' strand in the direction of translocation. Using DNA constructs with two neutral-backbone segments separated by a single charged base pair, we deduce that SpoIIIE's step size is 2 bp. Finally, experiments with non-hydrolyzable ATP analogs suggest that SpoIIIE can operate with non-consecutive inactive subunits. We propose a two-subunit escort translocation mechanism that is strict enough to enable SpoIIIE to track one DNA strand, yet sufficiently compliant to permit the motor to bypass inactive subunits without arrest. We speculate that such a flexible mechanism arose for motors that, like SpoIIIE, constitute functional bottlenecks where the inactivation of even a single motor can be lethal for the cell

Mechanochemical coupling and bi-phasic force-velocity dependence in the ultra-fast ring ATPase SpoIIIE.

Multi-subunit ring-shaped ATPases are molecular motors that harness chemical free energy to perform vital mechanical tasks such as polypeptide translocation, DNA unwinding, and chromosome segregation. Previously we reported the intersubunit coordination and stepping behavior of the hexameric ring-shaped ATPase SpoIIIE (Liu et al., 2015). Here we use optical tweezers to characterize the motor's mechanochemistry. Analysis of the motor response to external force at various nucleotide concentrations identifies phosphate release as the likely force-generating step. Analysis of SpoIIIE pausing indicates that pauses are off-pathway events. Characterization of SpoIIIE slipping behavior reveals that individual motor subunits engage DNA upon ATP binding. Furthermore, we find that SpoIIIE's velocity exhibits an intriguing bi-phasic dependence on force. We hypothesize that this behavior is an adaptation of ultra-fast motors tasked with translocating DNA from which they must also remove DNA-bound protein roadblocks. Based on these results, we formulate a comprehensive mechanochemical model for SpoIIIE

Mechanochemical coupling and bi-phasic force-velocity dependence in the ultra-fast ring ATPase SpoIIIE.

Multi-subunit ring-shaped ATPases are molecular motors that harness chemical free energy to perform vital mechanical tasks such as polypeptide translocation, DNA unwinding, and chromosome segregation. Previously we reported the intersubunit coordination and stepping behavior of the hexameric ring-shaped ATPase SpoIIIE (Liu et al., 2015). Here we use optical tweezers to characterize the motor's mechanochemistry. Analysis of the motor response to external force at various nucleotide concentrations identifies phosphate release as the likely force-generating step. Analysis of SpoIIIE pausing indicates that pauses are off-pathway events. Characterization of SpoIIIE slipping behavior reveals that individual motor subunits engage DNA upon ATP binding. Furthermore, we find that SpoIIIE's velocity exhibits an intriguing bi-phasic dependence on force. We hypothesize that this behavior is an adaptation of ultra-fast motors tasked with translocating DNA from which they must also remove DNA-bound protein roadblocks. Based on these results, we formulate a comprehensive mechanochemical model for SpoIIIE