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 fusion1. The AAA+ ATPase motor cytoplasmic dynein regulates ciliary trafficking2, mitotic spindle formation3, and organelle transport4, and dissecting its precise functions has been challenging due to 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 signaling 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.

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) ε to the exclusion of Pol δ on the leading strand. Even if Pol δ assembles on the leading strand, Pol ε 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 ε and stabilizing Pol ε. Comparison of Pol ε and Pol δ on a lagging-strand model DNA reveals the opposite. Pol δ dominates over excess Pol ε on PCNA-primed ssDNA. Thus, PCNA strongly favors Pol δ over Pol ε on the lagging strand, but CMG over-rides and flips this balance in favor of Pol ε on the leading strand.

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 introductory chapter 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 with cell division to ensure that the genome replicates once per cell division cycle.

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 e 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 e for leading-strand synthesis, but to date a direct interaction between CMG and Pol e has not been demonstrated. While purifying CMG helicase overexpressed in yeast, we detected a functional complex between CMG and native Pol e. Using pure CMG and Pol e, we reconstituted a stable 15-subunit CMG-Pol e 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 e is inefficient in CMG-dependent replication, but addition of the Dpb2 protein subunit of Pol e, 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 e on the leading strand as part of a 15-subunit leading-strand holoenzyme we refer to as CMGE. Direct binding between Pol e and CMG provides an explanation for specific targeting of Pol e to the leading strand and provides clear mechanistic evidence for how strand asymmetry is maintained in eukaryotes.DNA replication | replication fork | helicase | polymerase | CMG

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 ␤-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.DNA repair ͉ DNA replication ͉ replication fork ͉ replisome sliding clamp C hromosomes are duplicated by replisome machines containing helicase, primase, and DNA polymerase activites (1). Replicative DNA polymerases are tethered to DNA by ringshaped sliding clamp proteins. The Escherichia coli polymerase III (Pol III) holoenzyme replicase contains 10 different subunits consisting of a clamp loader that binds 2 molecules of Pol III, each tethered to DNA by the ␤-sliding clamp for processive leading and lagging strand synthesis (2). Pol III also binds tightly to the hexameric DnaB helicase that encircles the lagging strand template. The Pol III-to-DnaB interaction is mediated by the -subunit within Pol III (3). This tight connection couples Pol III motion to DnaB unwinding and increases the rate of unwinding by DnaB helicase from a basal level of 35 bp/s to a rate in excess of 500 bp/s (3). These tight connections of Pol III to DnaB and the ␤-clamp provide the replisome with sufficient stability, in principle, to replicate the entire genome. In practice, however, replication of long chromosomal DNA is an uneven path with a variety of obstacles along the way, including protein blocks and damaged templates.In the presence of high levels of DNA damage, Ͼ40 gene products are induced by the SOS response that act to restore genomic integrity and assure cell survival (4). Among the SOS-induced gene products are 3 translesion DNA polymerases, Pols II, IV, and V (encoded by polB, dinB, and umuCD, respectively), that perform potentially mutagenic DNA synthesis across template lesions (5). Pols IV and V are members of the Y family of error-prone DNA polymerases; they lack a proofreading 3Ј-5Ј exonuclease (6, 7). Pol II is a B-family polymerase; it has high fidelity and contains proofreading activity (8). Pols II and IV are present during normal cell growth and may be involved in repairing low levels of DNA damage that occur routinely in the cell (9-11). Pol V, however, is closely associated with mutagenesis and is not detectable in cells under normal conditions (7,12,13).Pol II and Pol IV are among the first gen...

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

Replicative helicases in all cell types are hexameric rings that unwind DNA by steric exclusion in which the helicase encircles the tracking strand only and excludes the other strand from the ring. This mode of translocation allows helicases to bypass blocks on the strand that is excluded from the central channel. Unlike other replicative helicases, eukaryotic CMG helicase partially encircles duplex DNA at a forked junction and is stopped by a block on the non-tracking (lagging) strand. This report demonstrates that Mcm10, an essential replication protein unique to eukaryotes, binds CMG and greatly stimulates its helicase activity in vitro. Most significantly, Mcm10 enables CMG and the replisome to bypass blocks on the non-tracking DNA strand. We demonstrate that bypass occurs without displacement of the blocks and therefore Mcm10 must isomerize the CMG-DNA complex to achieve the bypass function.

In most cells, 100 -1000 Okazaki fragments are produced for each replicative DNA polymerase present in the cell. For fastgrowing 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 ␦ (pol ␦), functions with the proliferating cell nuclear antigen (PCNA) sliding clamp. In this report, Saccharomyces cerevisiae pol ␦ is examined on model substrates to gain insight into the mechanism of lagging strand replication in eukaryotes. Surprisingly, we find pol ␦ 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 ␦ 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 ␦ ejects from PCNA upon extending a DNA template to completion and running into the downstream duplex. The released pol ␦ 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 ␦ heterotrimer.Chromosome replication in eukaryotes utilizes both DNA polymerase (pol) 2 ⑀ and DNA pol ␦, which are thought to function on the leading and lagging strands of the replication fork, respectively (1, 2). During replication fork movement, discontinuous lagging strand fragments are initiated by RNA primers generated by the primase function of DNA polymerase ␣/primase (pol ␣/primase), and the primers are extended by DNA pol ␣ to a total length of 30 -35 nucleotides. Unlike pol ␣, pol ␦ and pol ⑀ both utilize PCNA, a ring-shaped sliding clamp that increases the processivity of these DNA polymerases, and the PCNA clamp is assembled onto DNA by the 5-subunit RFC clamp loader (3-7). In these respects the eukaryotic replication apparatus is similar to the bacterial replisome, which contains sliding clamps and a clamp loader, except the bacterial replisome utilizes two identical copies of a DNA polymerase (pol III) for the leading and lagging strands (8). Eukaryotes also contain a heterohexameric MCM 2-7 complex, which is thought to be the replicative helicase, analogous to the Escherichia coli DnaB homohexamer (9).Despite the similarities between bacterial and eukaryotic replisomes, they appear to diverge significantly in many other respects. For example, the bacterial clamp loader is tightly associated with the leading and lagging stand polymerases, whereas the RFC clamp loader lacks stabile interactions with either pol ␦ or pol ⑀. Furth...