Research Event

Event participants

23 April 2020

The actin cytoskeleton is extremely dynamic and supports diverse cellular functions in many physiological and pathological processes, including tumorigenesis. However, the mechanisms that regulate the actin-related protein 2/3 (ARP2/3) complex and thereby promote actin polymerization and organization in cancer cells are not well-understood. We previously implicated the proline-rich 11 (PRR11) protein in lung cancer development. In this study, using immunofluorescence staining, actin polymerization assays, and siRNA-mediated gene silencing, we uncovered that cytoplasmic PRR11 is involved in F-actin polymerization and organization. We found that dysregulation of PRR11 expression results in F-actin rearrangement and nuclear instability in non-small cell lung cancer cells. Results from molecular mechanistic experiments indicated that PRR11 associates with and recruits the ARP2/3 complex, facilitates F-actin polymerization, and thereby disrupts the F-actin cytoskeleton, leading to abnormal nuclear lamina assembly and chromatin reorganization. Inhibition of the ARP2/3 complex activity abolished irregular F-actin polymerization, lamina assembly, and chromatin reorganization due to PRR11 overexpression. Notably, experiments with truncated PRR11 variants revealed that PRR11 regulates F-actin through different regions. We found that deletion of either the N or C terminus of PRR11 abrogates its effects on F-actin polymerization and nuclear instability and that deletion of amino acid residues 100–184 or 100–200 strongly induces an F-actin structure called the actin comet tail, not observed with WT PRR11. Our findings indicate that cytoplasmic PRR11 plays an essential role in regulating F-actin assembly and nuclear stability by recruiting the ARP2/3 complex in human non-small cell lung carcinoma cells.

Knowledge of the molecular events in mitochondrial DNA (mtDNA) replication is crucial to understanding the origins of human disorders arising from mitochondrial dysfunction. Twinkle helicase is an essential component of mtDNA replication. Here, we employed atomic force microscopy imaging in air and liquids to visualize ring assembly, DNA binding, and unwinding activity of individual Twinkle hexamers at the single-molecule level. We observed that the Twinkle subunits self-assemble into hexamers and higher-order complexes that can switch between open and closed-ring configurations in the absence of DNA. Our analyses helped visualize Twinkle loading onto and unloading from DNA in an open-ringed configuration. They also revealed that closed-ring conformers bind and unwind several hundred base pairs of duplex DNA at an average rate of ∼240 bp/min. We found that the addition of mitochondrial single-stranded (ss) DNA–binding protein both influences the ways Twinkle loads onto defined DNA substrates and stabilizes the unwound ssDNA product, resulting in a ∼5-fold stimulation of the apparent DNA-unwinding rate. Mitochondrial ssDNA-binding protein also increased the estimated translocation processivity from 1750 to >9000 bp before helicase disassociation, suggesting that more than half of the mitochondrial genome could be unwound by Twinkle during a single DNA-binding event. The strategies used in this work provide a new platform to examine Twinkle disease variants and the core mtDNA replication machinery. They also offer an enhanced framework to investigate molecular mechanisms underlying deletion and depletion of the mitochondrial genome as observed in mitochondrial diseases.

Incorporation of ribonucleotides into DNA can severely diminish genome integrity. However, how ribonucleotides instigate DNA damage is poorly understood. In DNA, they can promote replication stress and genomic instability and have been implicated in several diseases. We report here the impact of the ribonucleotide rATP and of its naturally occurring damaged analog 1,N6-ethenoadenosine (1,N6-ϵrA) on translesion synthesis (TLS), mediated by human DNA polymerase η (hpol η), and on RNase H2–mediated incision. Mass spectral analysis revealed that 1,N6-ϵrA in DNA generates extensive frameshifts during TLS, which can lead to genomic instability. Moreover, steady-state kinetic analysis of the TLS process indicated that deoxypurines (i.e. dATP and dGTP) are inserted predominantly opposite 1,N6-ϵrA. We also show that hpol η acts as a reverse transcriptase in the presence of damaged ribonucleotide 1,N6-ϵrA but has poor RNA primer extension activities. Steady-state kinetic analysis of reverse transcription and RNA primer extension showed that hpol η favors the addition of dATP and dGTP opposite 1,N6-ϵrA. We also found that RNase H2 recognizes 1,N6-ϵrA but has limited incision activity across from this lesion, which can lead to the persistence of this detrimental DNA adduct. We conclude that the damaged and unrepaired ribonucleotide 1,N6-ϵrA in DNA exhibits mutagenic potential and can also alter the reading frame in an mRNA transcript because 1,N6-ϵrA is incompletely incised by RNase H2.

In bacteria, the restart of stalled DNA replication forks requires the DNA helicase PriA. PriA can recognize and remodel abandoned DNA replication forks, unwind DNA in the 3'-to-5' direction, and facilitate the loading of the helicase DnaB onto the DNA to restart replication. Single-stranded DNA–binding protein (SSB) is typically present at the abandoned forks, but it is unclear how SSB and PriA interact, although it has been shown that the two proteins interact both physically and functionally. Here, we used atomic force microscopy to visualize the interaction of PriA with DNA substrates with or without SSB. These experiments were done in the absence of ATP to delineate the substrate recognition pattern of PriA before its ATP-catalyzed DNA-unwinding reaction. These analyses revealed that in the absence of SSB, PriA binds preferentially to a fork substrate with a gap in the leading strand. Such a preference has not been observed for 5'- and 3'-tailed duplexes, suggesting that it is the fork structure that plays an essential role in PriA's selection of DNA substrates. Furthermore, we found that in the absence of SSB, PriA binds exclusively to the fork regions of the DNA substrates. In contrast, fork-bound SSB loads PriA onto the duplex DNA arms of forks, suggesting a remodeling of PriA by SSB. We also demonstrate that the remodeling of PriA requires a functional C-terminal domain of SSB. In summary, our atomic force microscopy analyses reveal key details in the interactions between PriA and stalled DNA replication forks with or without SSB.

The action mechanisms revealed by the biochemical and structural analyses of replicative and translesion synthesis (TLS) DNA polymerases (Pols) are retained in their cellular roles. In this regard, DNA polymerase θ differs from other Pols in that whereas purified Polθ misincorporates an A opposite 1,N6-ethenodeoxyadenosine (ϵdA) using an abasic-like mode, Polθ performs predominantly error-free TLS in human cells. To test the hypothesis that Polθ adopts a different mechanism for replicating through ϵdA in human cells than in the purified Pol, here we analyze the effects of mutations in the two highly conserved tyrosine residues, Tyr-2387 and Tyr-2391, in the Polθ active site. Our findings that these residues are indispensable for TLS by the purified Pol but are not required in human cells, as well as other findings, provide strong evidence that the Polθ active site is reconfigured in human cells to stabilize ϵdA in the syn conformation for Hoogsteen base pairing with the correct nucleotide. The evidence that a DNA polymerase can configure its active site entirely differently in human cells than in the purified Pol establishes a new paradigm for DNA polymerase function.

Histone H2B monoubiquitylation (H2Bub1) has central functions in multiple DNA-templated processes, including gene transcription, DNA repair, and replication. H2Bub1 also is required for the trans-histone regulation of H3K4 and H3K79 methylation. Although previous studies have elucidated the basic mechanisms that establish and remove H2Bub1, we have only an incomplete understanding of how H2Bub1 is regulated. We report here that the histone H4 basic patch regulates H2Bub1. Yeast cells with arginine-to-alanine mutations in the H4 basic patch (H42RA) exhibited a significant loss of global H2Bub1. H42RA mutant yeast strains also displayed chemotoxin sensitivities similar to, but less severe than, strains containing a complete loss of H2Bub1. We found that the H4 basic patch regulates H2Bub1 levels independently of interactions with chromatin remodelers and separately from its regulation of H3K79 methylation. To measure H2B ubiquitylation and deubiquitination kinetics in vivo, we used a rapid and reversible optogenetic tool, the light-inducible nuclear exporter, to control the subcellular location of the H2Bub1 E3 ligase, Bre1. The ability of Bre1 to ubiquitylate H2B was unaffected in the H42RA mutant. In contrast, H2Bub1 deubiquitination by SAGA-associated Ubp8, but not by Ubp10, increased in the H42RA mutant. Consistent with a function for the H4 basic patch in regulating SAGA deubiquitinase activity, we also detected increased SAGA-mediated histone acetylation in H4 basic patch mutants. Our findings uncover that the H4 basic patch has a regulatory function in SAGA-mediated histone modifications.

A discussion marking the 25th anniversary of the 1990 Immigration Act, where experts examinine the history of the legislation, how it was accomplished politically, and the stakeholders and issues that were critical to its passage.

With issues of immigration policy very much at the forefront of the 2016 elections, this annual conference offers policy and legal analysis on key immigration topics facing the country and the incoming administration and Congress in 2017.

The 13th annual Immigration Law & Policy Conference offered policy and legal analysis on key immigration topics, including: the election and the future of immigration policy; refugee resettlement in the United States; immigration detention and enforcement; and the impacts of the Supreme Court opinion in the important U.S. vs Texas case

A discussion featuring data on immigration trends and the agricultural workforce, and some of the adjustments that farm employers are making, including increased mechanization, improved wages and benefits, and the increased use of the H-2A program. University of California-Davis’s Phil Martin, along with researchers from the the U.S. Department of Agriculture, U.S. Department of Labor, present their findings on the foreign agricultural workforce in the United States, which is followed by comments from the President of Farmworker Justice on some of the policy implications.

As policymakers in a number of countries, the United States among them, debate limiting family-based immigration, this issue brief explores family-migration trends and policies in the United States, Australia, Canada, the United Kingdom, and several other European countries. Family admissions play a key role, even in countries that prioritize economic or other immigration streams.

Where can you find the most accurate statistics on some of the top immigration issues—ranging from the size of immigrant and emigrant populations, to humanitarian and labor flows, naturalization rates, enforcement statistics, remittances, and more? In this brief video, Jeanne Batalova, who manages the Migration Policy Institute’s Migration Data Hub, walks you through the Immigration Data Matters guide developed by MPI and the Population Reference Bureau.
 

Who is an immigrant? Does that status change if, for example, a foreigner marries a native-born resident or serves in his or her adopted country's military? This explainer answers basic questions about international migrants—who they are, their top destinations, where they come from, how they are counted, and more.

With thousands more migrants potentially traveling through the Western Balkans this year, this MPI Europe webinar explores the implications of the buttressed EU border on the bloc’s neighbors, including the issues of outsourcing migration control, EU support for addressing irregular migration in neighboring countries, and considerations for EU policymakers.

With thousands migrants potentially traveling through the Western Balkans this year, this MPI Europe webinar explores the implications of the buttressed EU border on the bloc’s neighbors, the migrants transiting these routes, and the local communities. Experts also explored how the European Union can support efforts to address irregular migration in neighboring countries, and what are the tradeoffs and considerations that policymakers must weigh. 

As the U.S. health-care system sags under the strain of the COVID-19 pandemic, health-care workers are not only on the frontlines fighting the virus, they are also some of the most at-risk individuals, making it more essential than ever to have sufficient numbers of qualified health professionals. While 1.5 million immigrants and refugees are already working in the U.S. health-care system, an additional 263,000 immigrants and refugees in the U.S. with health-related college degrees are working in low-skilled jobs or are out of work.