Assistant Professor, Chemical and Systems Biology
Honors & Awards
Postdoctoral Fellowship, Jane Coffin Childs Memorial Fund (2014-2017)
Outstanding Graduate Student Instructor Award, University of California, Berkeley (2008)
Silver Medal, International Physics Olympiad (2002)
Postdoctoral, Harvard Medical School, Biological Chemistry & Molecular Pharmacology (2019)
Ph.D., University of California, Berkeley, Physics (Biophysics) (2013)
M.A., University of California, Berkeley, Physics (Biophysics) (2012)
B.S., Massachusetts Institute of Technology, Physics (2007)
Current Research and Scholarly Interests
Research in my laboratory is aimed at understanding how eukaryotes replicate their DNA despite numerous challenges (collectively known as replication stress), and more generally – how eukaryotic cells safeguard genome integrity. Specifically, we are investigating: (i) mechanisms that regulate the activity of the replicative helicase during replication stress, (ii) mechanisms that control the inheritance of epigenetic information during replication, and (iii) mechanisms of ubiquitin-mediated regulation of genome maintenance. We utilize single-molecule microscopy to directly image fluorescently-labeled replication factors and track them in real time in Xenopus egg extracts. I developed this system as a postdoctoral fellow, and used it to monitor how the eukaryotic replicative helicase copes with DNA damage. We plan to further extend the capabilities of this platform to directly visualize other essential replication factors, nucleosomes, and regulatory post-translational modifications like ubiquitin chains. By elucidating molecular mechanisms responsible for maintaining genome stability, we aim to better understand the link between genome instability and cancer, and how these mechanisms can be harnessed to improve disease treatment.
Postdoctoral Faculty Sponsor
Doctoral Dissertation Advisor (AC)
Scott Berger, Luke Lynch
TRAIP is a master regulator of DNA interstrand crosslink repair.
2019; 567 (7747): 267–72
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 crosslink1; 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 recombination2. 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 findings3,4 establish TRAIP as a master regulator of CMG unloading and the response of the replisome to obstacles.
View details for DOI 10.1038/s41586-019-1002-0
View details for PubMedID 30842657
View details for PubMedCentralID PMC6417926
The CMG Helicase Bypasses DNA-Protein Cross-Links to Facilitate Their Repair.
2019; 176 (1-2): 167–81.e21
Covalent DNA-protein cross-links (DPCs) impede replication fork progression and threaten genome integrity. Using Xenopus egg extracts, we previously showed that replication fork collision with DPCs causes their proteolysis, followed by translesion DNA synthesis. We show here that when DPC proteolysis is blocked, the replicative DNA helicase CMG (CDC45, MCM2-7, GINS), which travels on the leading strand template, bypasses an intact leading strand DPC. Single-molecule imaging reveals that GINS does not dissociate from CMG during bypass and that CMG slows dramatically after bypass, likely due to uncoupling from the stalled leading strand. The DNA helicase RTEL1 facilitates bypass, apparently by generating single-stranded DNA beyond the DPC. The absence of RTEL1 impairs DPC proteolysis, suggesting that CMG must bypass the DPC to enable proteolysis. Our results suggest a mechanism that prevents inadvertent CMG destruction by DPC proteases, and they reveal CMG's remarkable capacity to overcome obstacles on its translocation strand.
View details for DOI 10.1016/j.cell.2018.10.053
View details for PubMedID 30595447
View details for PubMedCentralID PMC6475077
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.
View details for DOI 10.7554/eLife.32354
View details for PubMedID 29504934
View details for PubMedCentralID PMC5858925
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.
View details for DOI 10.7554/eLife.09224
View details for PubMedID 26452092
View details for PubMedCentralID PMC4728128
Single-Molecule Visualization of MCM2-7 DNA Loading: Seeing Is Believing.
2015; 161 (3): 429–30
The first event in the initiation of eukaryotic DNA replication is the recruitment of the MCM2-7 ATPase, the core of the replicative DNA helicase, to origins. Ticau et al. use single-molecule imaging to reveal how ORC, Cdc6, and Cdt1 cooperate to load MCM2-7 onto DNA, enabling bidirectional replication.
View details for DOI 10.1016/j.cell.2015.04.006
View details for PubMedID 25910200
Mechanical operation and intersubunit coordination of ring-shaped molecular motors: insights from single-molecule studies.
2014; 106 (9): 1844–58
Ring NTPases represent a large and diverse group of proteins that couple their nucleotide hydrolysis activity to a mechanical task involving force generation and some type of transport process in the cell. Because of their shape, these enzymes often operate as gates that separate distinct cellular compartments to control and regulate the passage of chemical species across them. In this manner, ions and small molecules are moved across membranes, biopolymer substrates are segregated between cells or moved into confined spaces, double-stranded nucleic acids are separated into single strands to provide access to the genetic information, and polypeptides are unfolded and processed for recycling. Here we review the recent advances in the characterization of these motors using single-molecule manipulation and detection approaches. We describe the various mechanisms by which ring motors convert chemical energy to mechanical force or torque and coordinate the activities of individual subunits that constitute the ring. We also examine how single-molecule studies have contributed to a better understanding of the structural elements involved in motor-substrate interaction, mechanochemical coupling, and intersubunit coordination. Finally, we discuss how these molecular motors tailor their operation-often through regulation by other cofactors-to suit their unique biological functions.
View details for DOI 10.1016/j.bpj.2014.03.029
View details for PubMedID 24806916
View details for PubMedCentralID PMC4017299
- Molecular watchdogs on genome patrol. eLife 2014; 3: e02854
A viral packaging motor varies its DNA rotation and step size to preserve subunit coordination as the capsid fills.
2014; 157 (3): 702–13
Multimeric, ring-shaped molecular motors rely on the coordinated action of their subunits to perform crucial biological functions. During these tasks, motors often change their operation in response to regulatory signals. Here, we investigate a viral packaging machine as it fills the capsid with DNA and encounters increasing internal pressure. We find that the motor rotates the DNA during packaging and that the rotation per base pair increases with filling. This change accompanies a reduction in the motor's step size. We propose that these adjustments preserve motor coordination by allowing one subunit to make periodic, specific, and regulatory contacts with the DNA. At high filling, we also observe the downregulation of the ATP-binding rate and the emergence of long-lived pauses, suggesting a throttling-down mechanism employed by the motor near the completion of packaging. This study illustrates how a biological motor adjusts its operation in response to changing conditions, while remaining highly coordinated.
View details for DOI 10.1016/j.cell.2014.02.034
View details for PubMedID 24766813
View details for PubMedCentralID PMC4003460
High degree of coordination and division of labor among subunits in a homomeric ring ATPase.
2012; 151 (5): 1017–28
Ring NTPases 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 a comprehensive mechanochemical characterization of a homomeric ring ATPase-the bacteriophage φ29 packaging motor-a homopentamer that translocates double-stranded DNA 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 homopentamer bind and hydrolyze ATP during each cycle, only four participate in translocation, whereas the remaining subunit plays an ATP-dependent regulatory role.
View details for DOI 10.1016/j.cell.2012.10.031
View details for PubMedID 23178121
View details for PubMedCentralID PMC3652982
ClpX(P) Generates Mechanical Force to Unfold and Translocate Its Protein Substrates
2011; 145 (3): 459-469
AAA(+) unfoldases denature and translocate polypeptides into associated peptidases. We report direct observations of mechanical, force-induced protein unfolding by the ClpX unfoldase from E. coli, alone, and in complex with the ClpP peptidase. ClpX hydrolyzes ATP to generate mechanical force and translocate polypeptides through its central pore. Threading is interrupted by pauses that are found to be off the main translocation pathway. ClpX's translocation velocity is force dependent, reaching a maximum of 80 aa/s near-zero force and vanishing at around 20 pN. ClpX takes 1, 2, or 3 nm steps, suggesting a fundamental step-size of 1 nm and a certain degree of intersubunit coordination. When ClpX encounters a folded protein, it either overcomes this mechanical barrier or slips on the polypeptide before making another unfolding attempt. Binding of ClpP decreases the slip probability and enhances the unfolding efficiency of ClpX. Under the action of ClpXP, GFP unravels cooperatively via a transient intermediate.
View details for DOI 10.1016/j.cell.2011.04.010
View details for Web of Science ID 000290022900014
View details for PubMedID 21529717