Brian Kelch

Every day we make enough DNA to go to the moon and back. So, all our DNA replication enzymes work fast, right? In our latest preprint, we show that a key enzyme, the clamp loader ATPase RFC, releases ADP much slower than it loads PCNA. What gives? (1/16) doi.org/10.1101/2025...

I’d also like to point out that Josh never picked up a pipette before joining my lab! His PhD is in Mech Eng and purely computational! Yet in a short time he mastered biochem such that he was developing badass assays and his grid prep skillz are copied by others around UMass. (15/16)

I’d also like to thank all coauthors for their work on this manuscript, including lab alumni Drs. Jacob Landeck and Xingchen Liu, grad student Krishna Anand, and high school intern Sasha Litvak; UMass Chan cryo-EM facility staff Drs. Song, Chang & Ouch; and you for reading! (16/16)

RFC is conceptually analogous to a GTPase, which has dedicated Nucleotide Exchange Factors and NTPase Activating Partners. What makes RFC unique is that its own substrates are the NEF (PCNA) and NAP (DNA). (13/16)

At a time when science funding is so precious, we want to acknowledge that our work is NIH NIGMS funded, @joshua.pajak.bsky.social is funded by an American Cancer Society postdoc fellowship, and our MD sims used resources provided by NSF @accessforci.bsky.social and UMass Chan SCI. (14/16)

Josh performed more MD sims to try to understand how PCNA could promote nucleotide exchange in RFC. His sims predict that the D/E interface weakly bridged by ADP can be pried open as D binds PCNA, accelerating release. (11/16)

Based on our data, we propose a model whereby fast ATP binding to some subunits readies RFC to bind PCNA, and then PCNA completes RFC’s nucleotide exchange en route to clamp loading. We propose this prevents futile ATP hydrolysis and helps ensure RFC binds PCNA before DNA. (12/16)

So, what catalyzes nucleotide exchange? Josh developed a FRET assay using a labeled ATP analog to monitor ATP binding to RFC. RFC alone binds some ATPs fast but then binds more ATP slowly, consistent with A&B (and maybe C) exchanging faster than D. PCNA speeds up these slow binding steps! (9/16)

Thus, “PCNA is a Nucleotide Exchange Factor for Clamp Loader ATPase Complex”! It only took us 10 posts to get to the title of the paper😉. But how does PCNA do this? (10/16)

Next, we wondered how the dynamics of RFC influence ADP release? So, Josh performed MD sims. His sims predict that ATP-interfaces are tighter than ADP ones, and that the B/C interface can open farthest, offering an explanation why this interface is apo in our cryo-EM reconstructions. (7/16)

To test how interface opening plays a role in ADP release, Josh performed tRAMD(doi.org/10.1021/acs....), which is a technique that predicts relative release rates. He found that the half-off rate for C or D is slower than A or B, consistent with his cryo-EM. (8/16)

We wanted to understand the functional relevance of why they hold ADP tightly, so we started looking for clues, perhaps the most fun part of science. (5/16)

First, we looked at how RFC subunits respond to nucleotide binding by comparing Josh’s apo subunits to our lab’s previous ATPgS-bound subunits and found that the B&C subunits’ lids rotate in opposite directions! We had never seen this in a AAA+ before (if you know of any, please let us know!) (6/16)

To get insights into RFC recycling, postdoc @joshuapajak.bsky.social determined the structure of RFC in the apo state. He got great overall resolution of the core RFC complex, but the AAA+ domain of the A subunit is missing, suggesting it is flexibly tethered when apo. (3/16)

Imagine our surprise when we saw that the C and D subunits were ADP-bound! Because he never added nucleotide, these ADPs must have co-purified with RFC over a 3 day prep, meaning that RFC releases ADP very slowly from these subunits. (4/16)

Recently, our lab showed how an ATP-bound RFC loads PCNA onto DNA during replication and repair (doi.org/10.7554/eLif...). But RFC loads PCNA extremely fast, so that when RFC has done its job once, how does it prep for the next round? (2/16)

Did you know that there is so much DNA packed inside a phage capsid that the pressure is 10X higher than in a bottle of champagne? In our latest preprint, we wondered how that is contained in a phage that lives at extremely high temperatures. /1 www.biorxiv.org/content/10.1...

Putting that together with decades of research into phage genome packaging, we propose a speculative model for how the Portal switches from DNA-pumping mode to Tail attachment mode, using the pressure-induced piston motion of Portal to displace the motor. /12

It’s been a long road, so massive props to everyone who worked on this project, including lab alum Emily Agnello, PhD, graduate students Julia Hobaugh and Rakeyah Ahsan, and Chen Xu, PhD and Kangkang Song, PhD at the fantastic UMass Chan CryoEM facility! /13

On the left, Bayfield et al. showed the pore through Portal in an early, immature capsid. A specialized motor pumps the genome into the capsid through this open, hydrophilic pore. Then, it constricts in the mature phage, becoming narrow and hydrophobic. /10

So how does the Portal know it’s time to close the pore? We got a clue by comparing the position of the Portal in capsids before and after DNA has been pumped in. The pressure from DNA pushes the Portal lower in the capsid aperture like a piston! /11

We know that the Neck of this phage is sufficient to hold its DNA inside the capsid because we also isolated phage that have broken tails, and therefore nothing left but the Neck to retain DNA. /8

In 2020, Ollie Bayfield and Fred Antson predicted that one component of the Neck complex, the Portal protein (dark blue), would be constricted to hold DNA in the capsid of this phage. And they were right! /9 Bayfield et al elifesciences.org/articles/55517

Instead of beefing up electrostatic or hydrophobic interactions between these proteins, the phage intertwines different subunits to create topological linkages. This is the same strategy used to make a wicker basket strong! /6

But the phage has another problem—it needs a channel through the Neck and Tail to inject its genome into the host when it infects, but it can’t shoot its shot until it finds the right host. How does it keep that channel closed? /7

With 105 protein chains and 5 DNA segments, across 4 different symmetries, this is the largest structure the Kelch Lab has ever built (2.4 MDa)! The proteins in pinks/reds are part of the capsid shell, and they form an aperture that the rest of the Neck sits in. /4

If this aperture were to dilate, it would allow the genome to explode out of the capsid and kill the phage! So how does the phage hold this aperture together at high temperatures, with tons of DNA pressing against it? /5

This phage not only lives in hot springs but also has the longest tail of any known phage. Here you see 3 viruses infecting their host, T. thermophilus. One has released its genome into the host, but two still have DNA-filled heads. /2

Postdoc @emma.sedivy.bsky.social determined the structure of the Neck complex that joins the capsid head to the tail. We only knew the identity of 4 out of 8 of the proteins in the Neck, but we were able to identify the remaining 4 using ModelAngelo by @kjamali.bsky.social. /3

rdcu.beTargeting BRCA1-deficient PARP inhibitor-resistant cells with nickases reveals nick resection as a cancer vulnerabilityNature Cancer - Whalen et al. report that increased DNA end resection in BRCA-deficient, PARP inhibitor-resistant cancers leads to increased sensitivity to DNA nicks, limiting tumor formation in...

I'm a little late to the game, but I'm happy to announce that our paper with my @umasschan.bsky.social colleagues in the Travis Thomson lab has now been published in @plosbiology.org! Thanks to all involved on this fun collaboration!

PLOS Biology@plosbiology.org

#Transposons are parasitic genetic elements that act as raw material for evolution of new functions. @briankelch.bsky.social &co show that the capsid of the #virus-derived retrotransposon Copia mediates synaptic plasticity at the #Drosophila neuromuscular junction 🧪 @plosbiology.org plos.io/3X71IDa

Brian Kelch reposted

Josh Dubnau

@joshdubnau.bsky.social

20 Feb 2025

Fun primer with @ForeverYHChang highlighting a lovely Travis Thomson lab paper! Did animals co-opt viral gag for intercellular (trans synaptic) communication? Or did viruses co-opt retrotransposon gags that animals had previously co-opted for intercellular use?

PLOS Biology@plosbiology.org

Yung-Heng Chang & @joshdubnau.bsky.social explore a @plosbiology.org study revealing that the #retrotransposon gag protein of Copia forms virus-like capsids that transfer its own RNA across the fly neuromuscular junction to regulate #synapse formation 🧪 Paper: plos.io/3X71IDa Primer: plos.io/4i6rzmC

Brian Kelch reposted

Cantorlab

@cantorlab.bsky.social

21 Jan 2025

Congratulations to Jenna Whalen on her Nature Cancer paper out today-a distinct take on nicks and their toxicity! rdcu.be/d60Td

Brian Kelch reposted

Elinor Karlsson

@elinork.bsky.social

2 Nov 2023

Looking for TT faculty job? At #ASHG23? We’re recruiting! We = Genomics and Comp Bio dept at UMass Chan Medical School in Worcester, MA (not Boston but near Boston) academicjobsonline.org/ajo/jobs/25641 pop genomics, imaging, stat. genetics, machine learning aka cool science w big data.

academicjobsonline.orgUniversity of Massachusetts Chan Medical School, Department of Genomics and Computational Biology Full service online faculty recruitment and application management system for academic institutions worldwide. We offer unique solutions tailored for academic communities.

Brian Kelch reposted

Jonathan Watts

@jwattsgroup.bsky.social

19 Sept 2024

Hey, senior PhD students working on RNA and moving toward leading independent labs: Apply for the Maraganore Early Independence Fellow program at the UMass Chan RNA Therapeutics Institute and come work with us! www.linkedin.com/posts/angela...

linkedin.comAngela Messmer-Blust on LinkedIn: #diversityinstem #underrepresentedinscience #rnabiology…I am unbelievably excited and proud to launch the RNA Therapeutic Institute's new Maraganore Early Independence Fellows Program at UMass Chan Medical School…

Brian Kelch reposted

Amir Mitchell

@amitchell.bsky.social

20 Sept 2023

Systems and synthetic biologists, we are recruiting at the department of Systems Biology at the @UMassChan. Position is open to faculty at all levels. http://academicjobsonline.org/ajo/jobs/25423

academicjobsonline.orgUniversity of Massachusetts Chan Medical School, Departme...Full service online faculty recruitment and application m...

Brian Kelch reposted

Sven Klumpe

@svenklumpe.bsky.social

24 Feb 2024

Over 4 years in the making, I am extremely happy to share our pre-print on the “In-cell structure and snapshots of copia retrotransposons in intact tissue”. We used cryo-ET to resolve the copia capsid structure to subnanometer resolution inside cells: www.biorxiv.org/content/10.1...

23 Feb 2024

Check out this new preprint from first author @svenklumpe.bsky.social and coworkers (primarily Plitsko and Beck labs at Max Planck) describing Copia retrotransposon capsids inside cells. Fig 3 is so dope. Look at that capsid array! Beautiful work! 😍🤩 www.biorxiv.org/content/10.1...

I'm back to do an in-depth dive into our most recent work up on biorxiv. Led by the Thomson Lab in the Neurobiology dept, w Yumeng Liu of the Kelch Lab. We find that the retrotransposon Copia forms capsids that antagonize Arc for controlling synaptic plasticity. /1 www.biorxiv.org/content/10.1...

This leads to a mechanistic hypothesis that Copia and Arc antagonize each others’ capsid assembly by competing for RNA. The functional relevance of Copia in synaptic dev also suggests that we might be watching Copia in process of ‘domestication’. /6

For details, plz read the preprint. This is a huge team effort primarily from the Thomson lab, with biochemical and structural work from twitterless Yumeng Liu from my lab. /7

We find that Copia requires RNA for proper assembly of the capsid structure. The structure shows pores that presumably allow small molecules to enter the capsid, presumably for reverse transcriptase activity. Arc lacks these pores. /4

Amazingly, knockdown of Copia results in increased bouton development and synaptic plasticity, the opposite of an Arc knockout. Copia and Arc are antagonizing each other. 5/

The Thomson lab previously showed that the retrovirus-derived Arc gene forms virus-like capsid particles in extracellular vesicles (EVs). Arc capsids package and carry RNA across a neuromuscular junction (NMJ) for regulating synaptic plasticity. /2 pubmed.ncbi.nlm.nih.gov/29328915/

Here, they report that the retrotransposon Copia forms capsids that are also packaged into EVs that transport across the NMJ. We determined the structure of these virus-like particles, which are larger than Arc capsids, presumably to bind bigger Copia RNA. /3