Lance D. Langston

Small-molecule inhibitors of the AAA+ ATPase motor cytoplasmic dynein

The conversion of chemical energy into mechanical force by AAA+ (ATPases associated with diverse cellular activities) ATPases is integral to cellular processes, including DNA replication, protein unfolding, cargo transport and membrane fusion. The AAA+ ATPase motor cytoplasmic dynein regulates ciliary trafficking, mitotic spindle formation and organelle transport, and dissecting its precise functions has been challenging because of its rapid timescale of action and the lack of cell-permeable, chemical modulators. Here we describe the discovery of ciliobrevins, the first specific small-molecule antagonists of cytoplasmic dynein. Ciliobrevins perturb protein trafficking within the primary cilium, leading to their malformation and Hedgehog signalling blockade. Ciliobrevins also prevent spindle pole focusing, kinetochore–microtubule attachment, melanosome aggregation and peroxisome motility in cultured cells. We further demonstrate the ability of ciliobrevins to block dynein-dependent microtubule gliding and ATPase activity in vitro. Ciliobrevins therefore will be useful reagents for studying cellular processes that require this microtubule motor and may guide the development of additional AAA+ ATPase superfamily inhibitors.

Principles and Concepts of DNA Replication in Bacteria, Archaea, and Eukarya

The accurate copying of genetic information in the double helix of DNA is essential for inheritance of traits that define the phenotype of cells and the organism. The core machineries that copy DNA are conserved in all three domains of life: bacteria, archaea, and eukaryotes. This article outlines the general nature of the DNA replication machinery, but also points out important and key differences. The most complex organisms, eukaryotes, have to coordinate the initiation of DNA replication from many origins in each genome and impose regulation that maintains genomic integrity, not only for the sake of each cell, but for the organism as a whole. In addition, DNA replication in eukaryotes needs to be coordinated with inheritance of chromatin, developmental patterning of tissues, and cell division to ensure that the genome replicates once per cell division cycle.

Mechanism of asymmetric polymerase assembly at the eukaryotic replication fork

Eukaryotes use distinct polymerases for leading- and lagging-strand replication, but how they target their respective strands is uncertain. We reconstituted Saccharomyces cerevisiae replication forks and found that CMG helicase selects polymerase (Pol) ɛ to the exclusion of Pol δ on the leading strand. Even if Pol δ assembles on the leading strand, Pol ɛ rapidly replaces it. Pol δ–PCNA is distributive with CMG, in contrast to its high stability on primed ssDNA. Hence CMG will not stabilize Pol δ, instead leaving the leading strand accessible for Pol ɛ and stabilizing Pol ɛ. Comparison of Pol ɛ and Pol δ on a lagging-strand model DNA reveals the opposite. Pol δ dominates over excess Pol ɛ on PCNA-primed ssDNA. Thus, PCNA strongly favors Pol δ over Pol ɛ on the lagging strand, but CMG over-rides and flips this balance in favor of Pol ɛ on the leading strand.

Translesion DNA polymerases remodel the replisome and alter the speed of the replicative helicase

All cells contain specialized translesion DNA polymerases that replicate past sites of DNA damage. We find that Escherichia coli translesion DNA polymerase II (Pol II) and polymerase IV (Pol IV) function with DnaB helicase and regulate its rate of unwinding, slowing it to as little as 1 bp/s. Furthermore, Pol II and Pol IV freely exchange with the polymerase III (Pol III) replicase on the β-clamp and function with DnaB helicase to form alternative replisomes, even before Pol III stalls at a lesion. DNA damage-induced levels of Pol II and Pol IV dominate the clamp, slowing the helicase and stably maintaining the architecture of the replication machinery while keeping the fork moving. We propose that these dynamic actions provide additional time for normal excision repair of lesions before the replication fork reaches them and also enable the appropriate translesion polymerase to sample each lesion as it is encountered.

CMG helicase and DNA polymerase ε form a functional 15-subunit holoenzyme for eukaryotic leading-strand DNA replication

Significance All cells must replicate their chromosomes prior to cell division. This process is carried out by a collection of proteins, known as the replisome, that act together to unwind the double helix and synthesize two new DNA strands complementary to the two parental strands. The details of replisome function have been worked out for bacteria but are much less well understood for eukaryotic cells. We have developed a system for studying eukaryotic replisome function in vitro using purified proteins. Using this system, we have identified a direct interaction between the component that unwinds the DNA, the CMG (Cdc45-MCM-GINS) helicase, and the component that replicates the leading strand, DNA polymerase ε, to form a large helicase–polymerase holoenzyme comprising 15 separate proteins. DNA replication in eukaryotes is asymmetric, with separate DNA polymerases (Pol) dedicated to bulk synthesis of the leading and lagging strands. Pol α/primase initiates primers on both strands that are extended by Pol ε on the leading strand and by Pol δ on the lagging strand. The CMG (Cdc45-MCM-GINS) helicase surrounds the leading strand and is proposed to recruit Pol ε for leading-strand synthesis, but to date a direct interaction between CMG and Pol ε has not been demonstrated. While purifying CMG helicase overexpressed in yeast, we detected a functional complex between CMG and native Pol ε. Using pure CMG and Pol ε, we reconstituted a stable 15-subunit CMG–Pol ε complex and showed that it is a functional polymerase–helicase on a model replication fork in vitro. On its own, the Pol2 catalytic subunit of Pol ε is inefficient in CMG-dependent replication, but addition of the Dpb2 protein subunit of Pol ε, known to bind the Psf1 protein subunit of CMG, allows stable synthesis with CMG. Dpb2 does not affect Pol δ function with CMG, and thus we propose that the connection between Dpb2 and CMG helps to stabilize Pol ε on the leading strand as part of a 15-subunit leading-strand holoenzyme we refer to as CMGE. Direct binding between Pol ε and CMG provides an explanation for specific targeting of Pol ε to the leading strand and provides clear mechanistic evidence for how strand asymmetry is maintained in eukaryotes.

WHITHER THE REPLISOME: EMERGING PERSPECTIVES ON THE DYNAMIC NATURE OF THE DNA REPLICATION MACHINERY

Replisomes were originally thought to be multi-protein machines with a stabile and defined structure during replication. Discovery that replisomes repeatedly discard sliding clamps and assemble a new clamp to start each Okazaki fragment provided the first hint that the replisome structure changes during replication. Recent studies reveal that the replisome is more dynamic than ever thought possible. Replisomes can utilize many different polymerases; the helicase is regulated to travel at widely different speeds; leading and lagging strands need not always act in a coupled fashion with DNA loops; and the replication fork does not always exhibit semi-discontinuous replication. We review some of these findings here and discuss their implications for cell physiology as well as enzyme mechanism.

Reconstitution of a eukaryotic replisome reveals suppression mechanisms that define leading/lagging strand operation

We have reconstituted a eukaryotic leading/lagging strand replisome comprising 31 distinct polypeptides. This study identifies a process unprecedented in bacterial replisomes. While bacteria and phage simply recruit polymerases to the fork, we find that suppression mechanisms are used to position the distinct eukaryotic polymerases on their respective strands. Hence, Pol ε is active with CMG on the leading strand, but it is unable to function on the lagging strand, even when Pol δ is not present. Conversely, Pol δ-PCNA is the only enzyme capable of extending Okazaki fragments in the presence of Pols ε and α. We have shown earlier that Pol δ-PCNA is suppressed on the leading strand with CMG (Georgescu et al., 2014). We propose that CMG, the 11-subunit helicase, is responsible for one or both of these suppression mechanisms that spatially control polymerase occupancy at the fork. DOI: http://dx.doi.org/10.7554/eLife.04988.001

DNA Polymerase δ Is Highly Processive with Proliferating Cell Nuclear Antigen and Undergoes Collision Release upon Completing DNA

In most cells, 100-1000 Okazaki fragments are produced for each replicative DNA polymerase present in the cell. For fast-growing cells, this necessitates rapid recycling of DNA polymerase on the lagging strand. Bacteria produce long Okazaki fragments (1-2 kb) and utilize a highly processive DNA polymerase III (pol III), which is held to DNA by a circular sliding clamp. In contrast, Okazaki fragments in eukaryotes are quite short, 100-250 bp, and thus the eukaryotic lagging strand polymerase does not require a high degree of processivity. The lagging strand polymerase in eukaryotes, polymerase δ (pol δ), functions with the proliferating cell nuclear antigen (PCNA) sliding clamp. In this report, Saccharomyces cerevisiae pol δ is examined on model substrates to gain insight into the mechanism of lagging strand replication in eukaryotes. Surprisingly, we find pol δ is highly processive with PCNA, over at least 5 kb, on Replication Protein A (RPA)-coated primed single strand DNA. The high processivity of pol δ observed in this report contrasts with its role in synthesis of short lagging strand fragments, which require it to rapidly dissociate from DNA at the end of each Okazaki fragment. We find that this dilemma is solved by a “collision release” process in which pol δ ejects from PCNA upon extending a DNA template to completion and running into the downstream duplex. The released pol δ transfers to a new primed site, provided the new site contains a PCNA clamp. Additional results indicate that the collision release mechanism is intrinsic to the pol3/pol31 subunits of the pol δ heterotrimer.

A proposal: Evolution of PCNA's role as a marker of newly replicated DNA

Processivity clamps that hold DNA polymerases to DNA for processivity were the first proteins known to encircle the DNA duplex. At the time, polymerase processivity was thought to be the only function of ring shaped processivity clamps. But studies from many laboratories have identified numerous proteins that bind and function with sliding clamps. Among these processes are mismatch repair and nucleosome assembly. Interestingly, there exist polymerases that are highly processive and do not require clamps. Hence, DNA polymerase processivity does not intrinsically require that sliding clamps evolved for this purpose. We propose that polymerases evolved to require clamps as a way of ensuring that clamps are deposited on newly replicated DNA. These clamps are then used on the newly replicated daughter strands, for processes important to genomic integrity, such as mismatch repair and the assembly of nucleosomes to maintain epigenetic states of replicating cells during development.

Intrachromosomal tandem duplication and repeat expansion during attempts to inactivate the subtelomeric essential gene GSH1 in Leishmania

Gamma-glutamylcysteine synthetase encoded by GSH1 is the rate-limiting enzyme in the biosynthesis of glutathione and trypanothione in Leishmania. Attempts to generate GSH1 null mutants by gene disruption failed in Leishmania infantum. Removal of even a single allele invariably led to the generation of an extra copy of GSH1, maintaining two intact wild-type alleles. In the second and even third round of inactivation, the markers integrated at the homologous locus but always preserved two intact copies of GSH1. We probed into the mechanism of GSH1 duplication. GSH1 is subtelomeric on chromosome 18 and Southern blot analysis indicated that a 10-kb fragment flanked by 466-bp direct repeated sequences was duplicated in tandem on the same chromosomal allele each time GSH1 was targeted. Polymerase chain reaction analysis and sequencing confirmed the generation of novel junctions created at the level of the 466-bp repeats consequent to locus duplication. In loss of heterozygosity attempts, the same repeated sequences were utilized for generating extrachromosomal circular amplicons. Our results are consistent with break-induced replication as a mechanism for the generation of this regional polyploidy to compensate for the inactivation of an essential gene. This chromosomal repeat expansion through repeated sequences could be implicated in locus duplication in Leishmania.