DNA polymerase ζ has robust reverse transcriptase activity relative to other cellular DNA polymerases.

Cell biology and genetic studies have demonstrated that DNA double-strand break (DSB) repair can be performed using an RNA transcript that spans the site of the DNA break as a template for repair. This type of DSB repair requires a reverse transcriptase to convert an RNA sequence into DNA to facilitate repair of the break, rather than copying from a DNA template as in canonical DSB repair. Translesion synthesis (TLS) DNA polymerases (Pol) are often more promiscuous than DNA Pols, raising the notion that reverse transcription could be performed by a TLS Pol.

DNA polymerase ζ is a robust reverse transcriptase.

Cell biology and genetic studies have demonstrated that DNA double strand break (DSB) repair can be performed using an RNA transcript that spans the site of the DNA break as a template for repair. This type of DSB repair requires a reverse transcriptase to convert an RNA sequence into DNA to facilitate repair of the break, rather than copying from a DNA template as in canonical DSB repair. Translesion synthesis (TLS) DNA polymerases (Pol) are often more promiscuous than DNA Pols, raising the notion that reverse transcription could be performed by a TLS Pol.

Structures of the human leading strand Polε-PCNA holoenzyme.

In eukaryotes, the leading strand DNA is synthesized by Polε and the lagging strand by Polδ. These replicative polymerases have higher processivity when paired with the DNA clamp PCNA. While the structure of the yeast Polε catalytic domain has been determined, how Polε interacts with PCNA is unknown in any eukaryote, human or yeast.

Mechanism of PCNA loading by Ctf18-RFC for leading-strand DNA synthesis.

The proliferating cell nuclear antigen (PCNA) clamp encircles DNA to hold DNA polymerases (Pols) to DNA for processivity. The Ctf18-RFC PCNA loader, a replication factor C (RFC) variant, is specific to the leading-strand Pol (Polε). We reveal here the underlying mechanism of Ctf18-RFC specificity to Polε using cryo-electron microscopy and biochemical studies.

Cryo-EM reveals a nearly complete PCNA loading process and unique features of the human alternative clamp loader CTF18-RFC.

The DNA sliding clamp PCNA is a multipurpose platform for DNA polymerases and many other proteins involved in DNA metabolism. The topologically closed PCNA ring needs to be cracked open and loaded onto DNA by a clamp loader, e.g.

Structure of the PCNA unloader Elg1-RFC.

During DNA replication, the proliferating cell nuclear antigen (PCNA) clamps are loaded onto primed sites for each Okazaki fragment synthesis by the AAA heteropentamer replication factor C (RFC). PCNA encircling duplex DNA is quite stable and is removed from DNA by the dedicated clamp unloader Elg1-RFC. Here, we show the cryo-EM structure of Elg1-RFC in various states with PCNA.

Structures of 9-1-1 DNA checkpoint clamp loading at gaps from start to finish and ramification on biology.

Rad24-RFC (replication factor C) loads the 9-1-1 checkpoint clamp onto the recessed 5' ends by binding a 5' DNA at an external surface site and threading the 3' single-stranded DNA (ssDNA) into 9-1-1. We find here that Rad24-RFC loads 9-1-1 onto DNA gaps in preference to a recessed 5' end, thus presumably leaving 9-1-1 on duplex 3' ss/double-stranded DNA (dsDNA) after Rad24-RFC ejects from DNA. We captured five Rad24-RFC-9-1-1 loading intermediates using a 10-nt gap DNA.

Molecular choreography of primer synthesis by the eukaryotic Pol α-primase.

The eukaryotic polymerase α (Pol α) synthesizes an RNA-DNA hybrid primer of 20-30 nucleotides. Pol α is composed of Pol1, Pol12, Primase 1 (Pri1), and Pri2. Pol1 and Pri1 contain the DNA polymerase and RNA primase activities, respectively.

Structures of 9-1-1 DNA checkpoint clamp loading at gaps from start to finish and ramification to biology.

Recent structural studies show the Rad24-RFC loads the 9-1-1 checkpoint clamp onto a recessed 5' end by binding the 5' DNA on Rad24 at an external surface site and threading the 3' ssDNA into the well-established internal chamber and into 9-1-1. We find here that Rad24-RFC loads 9-1-1 onto DNA gaps in preference to a recessed 5' DNA end, thus presumably leaving 9-1-1 on a 3' ss/ds DNA after Rad24-RFC ejects from the 5' gap end and may explain reports of 9-1-1 directly functioning in DNA repair with various TLS polymerases, in addition to signaling the ATR kinase. To gain a deeper understanding of 9-1-1 loading at gaps we report high-resolution structures of Rad24-RFC during loading of 9-1-1 onto 10-nt and 5-nt gapped DNAs.

Molecular choreography of primer synthesis by the eukaryotic Pol α-primase.

The eukaryotic polymerase α (Pol α) is a dual-function DNA polymerase/primase complex that synthesizes an RNA-DNA hybrid primer of 20-30 nucleotides for DNA replication. Pol α is composed of Pol1, Pol12, Primase 1 (Pri1), and Pri2, with Pol1 and Pri1 containing the DNA polymerase activity and RNA primase activity, respectively, whereas Pol12 and Pri2 serve a structural role. It has been unclear how Pol α hands over an RNA primer made by Pri1 to Pol1 for DNA primer extension, and how the primer length is defined, perhaps due to the difficulty in studying the highly mobile structure.

SV40 T-antigen uses a DNA shearing mechanism to initiate origin unwinding.

Duplication of DNA genomes requires unwinding of the double-strand (ds) DNA so that each single strand (ss) can be copied by a DNA polymerase. The genomes of eukaryotic cells are unwound by two ring-shaped hexameric helicases that initially encircle dsDNA but transition to ssDNA for function as replicative helicases. How the duplex is initially unwound, and the role of the two helicases in this process, is poorly understood.

Smc5/6's multifaceted DNA binding capacities stabilize branched DNA structures.

Smc5/6 is an evolutionarily conserved SMC complex with roles in DNA replication and repair, as well as in viral DNA restriction. Understanding its multiple functions has been hampered by a lack of mechanistic studies on how the Smc5/6 complex associates with different types of DNA. Here we address this question by simultaneously visualizing the behavior of Smc5/6 on three types of DNA, namely double-stranded (ds) DNA, single-stranded (ss) DNA, and junction DNA formed by juxtaposed ss- and dsDNA, using correlative single-molecule fluorescence and force microscopy.

Origin recognition complex harbors an intrinsic nucleosome remodeling activity.

Eukaryotic DNA replication is initiated at multiple chromosomal sites known as origins of replication that are specifically recognized by the origin recognition complex (ORC) containing multiple ATPase sites. In budding yeast, ORC binds to specific DNA sequences known as autonomously replicating sequences (ARSs) that are mostly nucleosome depleted. However, nucleosomes may still inhibit the licensing of some origins by occluding ORC binding and subsequent MCM helicase loading.

Nucleosome-directed replication origin licensing independent of a consensus DNA sequence.

The numerous enzymes and cofactors involved in eukaryotic DNA replication are conserved from yeast to human, and the budding yeast Saccharomyces cerevisiae (S.c.) has been a useful model organism for these studies.

Cryo-EM structures reveal that RFC recognizes both the 3'- and 5'-DNA ends to load PCNA onto gaps for DNA repair.

RFC uses ATP to assemble PCNA onto primed sites for replicative DNA polymerases δ and ε. The RFC pentamer forms a central chamber that binds 3' ss/ds DNA junctions to load PCNA onto DNA during replication. We show here five structures that identify a second DNA binding site in RFC that binds a 5' duplex.

DNA is loaded through the 9-1-1 DNA checkpoint clamp in the opposite direction of the PCNA clamp.

The 9-1-1 DNA checkpoint clamp is loaded onto 5'-recessed DNA to activate the DNA damage checkpoint that arrests the cell cycle. The 9-1-1 clamp is a heterotrimeric ring that is loaded in Saccharomyces cerevisiae by Rad24-RFC (hRAD17-RFC), an alternate clamp loader in which Rad24 replaces Rfc1 in the RFC1-5 clamp loader of proliferating cell nuclear antigen (PCNA). The 9-1-1 clamp loading mechanism has been a mystery, because, unlike RFC, which loads PCNA onto a 3'-recessed junction, Rad24-RFC loads the 9-1-1 ring onto a 5'-recessed DNA junction.

CMG helicase can use ATPγS to unwind DNA: Implications for the rate-limiting step in the reaction mechanism.

The adenosine triphosphate (ATP) analog ATPγS often greatly slows or prevents enzymatic ATP hydrolysis. The eukaryotic CMG (Cdc45, Mcm2 to 7, GINS) replicative helicase is presumed unable to hydrolyze ATPγS and thus unable to perform DNA unwinding, as documented for certain other helicases. Consequently, ATPγS is often used to "preload" CMG onto forked DNA substrates without unwinding before adding ATP to initiate helicase activity.

Allosteric communication in DNA polymerase clamp loaders relies on a critical hydrogen-bonded junction.

Clamp loaders are AAA+ ATPases that load sliding clamps onto DNA. We mapped the mutational sensitivity of the T4 bacteriophage sliding clamp and clamp loader by deep mutagenesis, and found that residues not involved in catalysis or binding display remarkable tolerance to mutation. An exception is a glutamine residue in the AAA+ module (Gln 118) that is not located at a catalytic or interfacial site.

Anatomy of a twin DNA replication factory.

The replication of DNA in chromosomes is initiated at sequences called origins at which two replisome machines are assembled at replication forks that move in opposite directions. Interestingly, in vivo studies observe that the two replication forks remain fastened together, often referred to as a replication factory. Replication factories containing two replisomes are well documented in cellular studies of bacteria (Escherichia coli and Bacillus subtilis) and the eukaryote, Saccharomyces cerevisiae.

The DNA Replication Machine: Structure and Dynamic Function.

In all cell types, a multi-protein machinery is required to accurately duplicate the large duplex DNA genome. This central life process requires five core replisome factors in all cellular life forms studied thus far. Unexpectedly, three of the five core replisome factors have no common ancestor between bacteria and eukaryotes.

Structure of eukaryotic DNA polymerase δ bound to the PCNA clamp while encircling DNA.

The DNA polymerase (Pol) δ of (S.c.) is composed of the catalytic subunit Pol3 along with two regulatory subunits, Pol31 and Pol32.

Replisome bypass of a protein-based R-loop block by Pif1.

Efficient and faithful replication of the genome is essential to maintain genome stability. Replication is carried out by a multiprotein complex called the replisome, which encounters numerous obstacles to its progression. Failure to bypass these obstacles results in genome instability and may facilitate errors leading to disease.