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.

Cas12a (Cpf1) is an RNA-guided endonuclease in the bacterial type V-A CRISPR-Cas anti-phage immune system that can be repurposed for genome editing. Cas12a can bind and cut dsDNA targets with high specificity in vivo, making it an ideal candidate for expanding the arsenal of enzymes used in precise genome editing. However, this reported high specificity contradicts Cas12a's natural role as an immune effector against rapidly evolving phages. Here, we employed high-throughput in vitro cleavage assays to determine and compare the native cleavage specificities and activities of three different natural Cas12a orthologs (FnCas12a, LbCas12a, and AsCas12a). Surprisingly, we observed pervasive sequence-specific nicking of randomized target libraries, with strong nicking of DNA sequences containing up to four mismatches in the Cas12a-targeted DNA-RNA hybrid sequences. We also found that these nicking and cleavage activities depend on mismatch type and position and vary with Cas12a ortholog and CRISPR RNA sequence. Our analysis further revealed robust nonspecific nicking of dsDNA when Cas12a is activated by binding to a target DNA. Together, our findings reveal that Cas12a has multiple nicking activities against dsDNA substrates and that these activities vary among different Cas12a orthologs.

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.

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

With immigration increasingly visible in the news and the political space in the United States and internationally, getting access to accurate, high-quality data is essential to understand immigration’s demographic effects and impacts on the economy, education and labor systems, and communities. This event marks the release of the Immigration Data Matters guide.

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.

What President Trump calls a border crisis is in fact a crisis in the asylum system—one worsened at every turn by his administration’s harsh policies and rhetoric. Rather than spend $5.7 billion on a wall, it would be far more effective to use the money to retool an overwhelmed asylum system, adapt outmatched border enforcement infrastructure to respond to the changing composition of arrivals, and work cooperatively with Mexico to tackle the factors propelling Central Americans to flee.

Though a faceoff between the U.S. executive and legislative branches is now in the courts, with President Trump's decision to declare a national emergency so he can allocate more money for construction of a border wall, a less-noted dispute has been taking place over the Department of Homeland Security's decision to add thousands more immigration detention beds than Congress provides annually, as this article explains.

Even as the 1.8 million number swirls in the discussion of how many DREAMers would be placed on a path to citizenship, proposals debated in the Senate in February 2018 would have resulted in the legalization of smaller numbers, as this commentary explains. It offers estimates of potential beneficiaries of several Senate proposals, including one backed by the White House, and analysis of key criteria.

The House is set to vote on two bills that would largely dismantle the U.S. asylum system at the southern border by significantly narrowing grounds to apply for asylum, eliminating protections for the vast majority of unaccompanied minors, and unilaterally declaring Mexico a safe third country. The result would be a sharp reduction in the number of people permitted to seek humanitarian protection, as this commentary explains.

In exchange for resolving the status of DREAMers, the White House and its congressional allies are demanding billions of dollars for a border wall and additional enforcement, sharp limits on asylum, cuts to legal immigration, and more. But what would the two bills expected to be voted on by the House do in terms of extending temporary or permanent status to DREAMers? This commentary offers estimates.

A report release and presentation of first-ever U.S. estimates on the actual economic costs of skill underutilization for immigrants, their families, and the U.S. economy, in terms of forgone earnings and unrealized federal, state, and local taxes.

During its first year, the Trump administration methodically put in place a series of bureaucratic barriers that could significantly reduce opportunities for foreigners to come to the United States legally. Among the actions taken during 2017: Imposition of a much-challenged travel ban suspending the entry of nationals from certain Muslim-majority countries, cuts to refugee admissions, and increased scrutiny for visa applicants.

At this release of an updated version of the popular Immigration Data Matters guide, presenters discuss how to navigate and access the increasing number of data sources on immigration and immigrants in the United States and internationally. 

Award winners for the inaugural year of the E Pluribus Unum Prizes program were honored at a reception at the Library of Congress in Washington, DC in 2009.

This awards ceremony, honoring the 2011 recipients of the E Pluribus Unum Prizes — a national awards program for exceptional immigrant integration initiatives — featured panel discussions with the awardees and federal officials and remarks by White House Director of Intergovernmental Affairs Cecilia Muñoz and Assistant Secretary of Education Brenda Dann-Messier.

This awards ceremony, honoring the 2011 recipients of the E Pluribus Unum Prizes -- a national awards program for exceptional immigrant integration initiatives -- featured panel discussions with the awardees and federal officials and remarks by White House Director of Intergovernmental Affairs Cecilia Muñoz and Assistant Secretary of Education Brenda Dann-Messier.

This awards ceremony honored the 2013 winners of the E Pluribus Unum Prizes, which provides $50,000 prizes to exceptional U.S. immigrant integration initiatives. The awardees took part in a panel discussion with White House and state officials, followed by remarks from Congressman Luis Gutierrez and U.S. Senator Amy Klobuchar.

Marking the release of an MPI report, this webinar examines what the growing intersection between U.S. immigration and child welfare systems means for protection agencies. Speakers also discuss promising child welfare policies and agency approaches to address the needs of children of immigrants and their families amid demographic change and rising immigration enforcement.

The Trump administration’s decision to terminate the Deferred Action for Childhood Arrivals (DACA) deportation-relief program launched in 2012 has sparked new urgency to find a longer-term fix for "DREAMers," the unauthorized immigrants brought to the United States as children. This Policy Beat article examines movement in the courts and in Congress on the DREAM Act and similar proposals, exploring likely paths forward.

In a year when immigration has been prominent in the headlines, the 14th annual Immigration Law and Policy Conference provides timely policy and legal analysis, with expert discussion covering the shifting immigration policy priorities under a new administration, including changes in immigration enforcement, border enforcement, refugee resettlement, the treatment of unaccompanied minors and their families, Temporary Protected Status, and DACA, along with the responses from the judicial and congressional branches, various stakeholders, and state and local governments. 

In its first year, the Trump administration moved to deliver on some of Donald Trump’s campaign promises on immigration, including ramping up enforcement in the U.S. interior and ending the Deferred Action for Childhood Arrivals (DACA) program. The administration also announced the termination of Temporary Protected Status (TPS) for nationals of some countries. This article explores some of the top policy changes.