Roxana Georgescu - Academia.edu (original) (raw)

Papers by Roxana Georgescu

Proceedings of the National Academy of Sciences

Duplication of DNA genomes requires unwinding of the double-strand (ds) DNA so that each single s... more 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. We recently described an initiation mechanism for eukaryotes in which the two helicases are directed inward toward one another and shear the duplex open by pulling on opposite strands of the duplex while encircling dsDNA [L. D. Langston, M. E. O'Donnell, eLife 8 , e46515 (2019)]. Two head-to-head T-Antigen helicases are long known to be loaded at the SV40 origin. We show here that T-Antigen tracks head (N-tier) first on ssDNA, opposite the direction proposed for decades. We also find that SV40 T-Antigen tracks directionally while encircling dsDNA and mainly ...

eLife

RFC uses ATP to assemble PCNA onto primed sites for replicative DNA polymerases δ and ε. The RFC ... more 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. This 5′ DNA site is located between the N-terminal BRCT domain and AAA+ module of the large Rfc1 subunit. Our structures reveal ideal binding to a 7-nt gap, which includes 2 bp unwound by the clamp loader. Biochemical studies show enhanced binding to 5 and 10 nt gaps, consistent with the structural results. Because both 3′ and 5′ ends are present at a ssDNA gap, we propose that the 5′ site facilitates RFC’s PCNA loading activity at a DNA damage-induced gap to recruit gap-filling polymerases. These findings are consistent with genetic studies showing that base excision repair of gaps greater than 1 base requires PCNA and involves the 5′ DNA binding domain of Rfc1. We further observe ...

Nature Communications, 2020

The eukaryotic leading strand DNA polymerase (Pol) ε contains 4 subunits, Pol2, Dpb2, Dpb3 and Dp... more The eukaryotic leading strand DNA polymerase (Pol) ε contains 4 subunits, Pol2, Dpb2, Dpb3 and Dpb4. Pol2 is a fusion of two B-family Pols; the N-terminal Pol module is catalytic and the C-terminal Pol module is non-catalytic. Despite extensive efforts, there is no atomic structure for Pol ε holoenzyme, critical to understanding how DNA synthesis is coordinated with unwinding and the DNA path through the CMG helicase-Pol ε-PCNA clamp. We show here a 3.5-Å cryo-EM structure of yeast Pol ε revealing that the Dpb3–Dpb4 subunits bridge the two DNA Pol modules of Pol2, holding them rigid. This information enabled an atomic model of the leading strand replisome. Interestingly, the model suggests that an OB fold in Dbp2 directs leading ssDNA from CMG to the Pol ε active site. These results complete the DNA path from entry of parental DNA into CMG to exit of daughter DNA from PCNA.

ABSTRACTOur earlier study demonstrated that head-to-head CMGs that encircle duplex DNA and track ... more ABSTRACTOur earlier study demonstrated that head-to-head CMGs that encircle duplex DNA and track inward at origins, melt double-strand (ds) DNA while encircling the duplex by pulling on opposite strands and shearing DNA apart (Langston and O’Donnell (2019) eLife9, e46515). We show here that increasing the methylphosphonate neutral DNA from 10 nucleotides in the previous report, to 20 nucleotides, reveals that CMG encircling duplex DNA only interacts with the tracking strand compared to the non-tracking strand. This significantly enhances support that CMG tracks on duplex DNA by binding only one strand. Furthermore, EMSA assays using AMPPNP to load CMG onto DNA shows a stoichiometry of only 2 CMGs on an origin mimic DNA, containing a 150 bp duplex with two 3 prime single-strand (ss) tails, one on each end, enabling assay of dsDNA unwinding by a shearing force produced by only two head-to-head CMGs. The use of non-hydrolysable AMPPNP enabled a preincubation for CMG binding the two 3 p...

The current view is that eukaryotic replisomes are independent. Here we show that Ctf4 tightly di... more The current view is that eukaryotic replisomes are independent. Here we show that Ctf4 tightly dimerizes CMG helicase, with an extensive interface involving Psf2, Cdc45, and Sld5. Interestingly, Ctf4 binds only one Pol α-primase. Thus, Ctf4 may have evolved as a trimer to organize two helicases and one Pol α-primase into a replication factory. In the 2CMG-Ctf43-1Pol α-primase factory model, the two CMGs nearly face each other, placing the two lagging strands toward the center and two leading strands out the sides. The single Pol α-primase is centrally located and may prime both sister replisomes. The Ctf4-coupled-sister replisome model is consistent with cellular microscopy studies revealing two sister forks of an origin remain attached and are pushed forward from a protein platform. The replication factory model may facilitate parental nucleosome transfer during replication.

Advances in experimental medicine and biology, 2017

Eukaryotic replication proteins are highly conserved, and thus study of Saccharomyces cerevisiae ... more Eukaryotic replication proteins are highly conserved, and thus study of Saccharomyces cerevisiae replication can inform about this central process in higher eukaryotes including humans. The S. cerevisiae replisome is a large and dynamic assembly comprised of ~50 proteins. The core of the replisome is composed of 31 different proteins including the 11-subunit CMG helicase; RFC clamp loader pentamer; PCNA cl& the heteroligomeric DNA polymerases ε, δ, and α-primase; and the RPA heterotrimeric single strand binding protein. Many additional protein factors either travel with or transiently associate with these replisome proteins at particular times during replication. In this chapter, we summarize several recent structural studies on the S. cerevisiae replisome and its subassemblies using single particle electron microscopy and X-ray crystallography. These recent structural studies have outlined the overall architecture of a core replisome subassembly and shed new light on the mechanism ...

Replicative helicases of all cell types are rings that unwind DNA by steric exclusion in which th... more Replicative helicases of all cell types are rings that unwind DNA by steric exclusion in which the helicase ring only encircles the tracking strand, excluding the other strand outside the ring. Steric exclusion mediated unwinding enables helicase rings to bypass blocks on the strand that is excluded from the central channel. Unlike other replicative helicases, eukaryotic CMG 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 enables the replisome to bypass blocks on the non-tracking strand, implying that Mcm10 isomerizes the CMG-DNA complex to position only one strand through the central channel. A similar CMG-DNA isomerization is needed at the origin for head-to-head CMGs to bypass one another during formation of bidirectional replication forks.

Proceedings of the National Academy of Sciences of the United States of America, Jan 31, 2017

The eukaryotic CMG (Cdc45, Mcm2-7, GINS) helicase consists of the Mcm2-7 hexameric ring along wit... more The eukaryotic CMG (Cdc45, Mcm2-7, GINS) helicase consists of the Mcm2-7 hexameric ring along with five accessory factors. The Mcm2-7 heterohexamer, like other hexameric helicases, is shaped like a ring with two tiers, an N-tier ring composed of the N-terminal domains, and a C-tier of C-terminal domains; the C-tier contains the motor. In principle, either tier could translocate ahead of the other during movement on DNA. We have used cryo-EM single-particle 3D reconstruction to solve the structure of CMG in complex with a DNA fork. The duplex stem penetrates into the central channel of the N-tier and the unwound leading single-strand DNA traverses the channel through the N-tier into the C-tier motor, 5'-3' through CMG. Therefore, the N-tier ring is pushed ahead by the C-tier ring during CMG translocation, opposite the currently accepted polarity. The polarity of the N-tier ahead of the C-tier places the leading Pol ε below CMG and Pol α-primase at the top of CMG at the replic...

Http Www Theses Fr, 1998

Le repliement de la thioredoxine se fait selon 5 etapes. La phase initiale tres rapide (<2. 5 ... more Le repliement de la thioredoxine se fait selon 5 etapes. La phase initiale tres rapide (<2. 5 ms) correspond essentiellement a une population heterogene comportant le feuillet. Le chemin qui mene de cet etat precoce vers l'etat final comporte 3 voies dont l'une mene directement vers l'etat natif ; les 2 autres convergent vers un etat intermediaire, dont la conversion en molecules natives est controlee par l'isomerisation transcis de la proline 76. Le clivage a l'arginine genere deux fragments desordonnes a l'etat isole mais aptes a se reassocier (k d=5. 10 - 8m). Le complexe garde la topologie / et l'empilement de la proteine native. L'assemblage est triphasique. La constante de vitesse de l'etape bimoleculaire est 1300 m - 1. S - 1. Comme dans le cas du repliement de la proteine non clivee, il existe une phase tres lente d'amplitude comparable et de meme constante de vitesse : 0,002 s - 1, liee a l'isomerisation trans-cis de la proline 76. Le fragment 1-73 existe sous deux formes a l'equilibre, dont une seule est competente pour l'association. L'isomerisation entre formes incompetente et competente est caracterisee par une constante de vitesse de 0,002 s - 1. Le fragment 1-73 competent s'associe indifferemment aux les deux formes p76-cis et p76-trans du fragment complementaire. Le complexe trans est majoritairement forme et subit l'isomerisation (k=0,002 s - 1) pour donner le complexe cis pseudo-natif. La transition de denaturation thermique des fragments isoles, est reversible non cooperative et revele des processus de denaturation a basse temperature. Les transitions de denaturation de la proteine intacte et du complexe sont cooperatives et reversibles et refletent la perte concertee des structures secondaire et tertiaire dans les deux cas. L'energie libre a 20\c et l'enthalpie de denaturation pour la proteine intacte et le complexe sont respectivement 9. 4 et 103 kcal/mol, et 10 et 116 kcal/mol. La concentration effective du complexe se situe au voisinage de 1 m.

Nature structural & molecular biology, Jan 8, 2016

The CMG helicase is composed of Cdc45, Mcm2-7 and GINS. Here we report the structure of the Sacch... more The CMG helicase is composed of Cdc45, Mcm2-7 and GINS. Here we report the structure of the Saccharomyces cerevisiae CMG, determined by cryo-EM at a resolution of 3.7-4.8 Å. The structure reveals that GINS and Cdc45 scaffold the N tier of the helicase while enabling motion of the AAA+ C tier. CMG exists in two alternating conformations, compact and extended, thus suggesting that the helicase moves like an inchworm. The N-terminal regions of Mcm2-7, braced by Cdc45-GINS, form a rigid platform upon which the AAA+ C domains make longitudinal motions, nodding up and down like an oil-rig pumpjack attached to a stable platform. The Mcm ring is remodeled in CMG relative to the inactive Mcm2-7 double hexamer. The Mcm5 winged-helix domain is inserted into the central channel, thus blocking entry of double-stranded DNA and supporting a steric-exclusion DNA-unwinding model.

Molecular Cell, 2003

strand must exhibit behavior that contrasts with such high processivity. The lagging strand is sy... more strand must exhibit behavior that contrasts with such high processivity. The lagging strand is synthesized as 1230 York Avenue New York, New York 10021 short 1-3 kb Okazaki fragments. Primase initiates each fragment by synthesis of a short RNA primer. As the polymerase extends the primer, it synthesizes DNA in the direction opposite fork movement even though it Summary travels with the fork due to its connection to both DnaB (through) and the leading polymerase (through the The E. coli replication machinery employs a ␤ clamp that tethers the polymerase to DNA, thus ensuring high shared clamp loader). These opposite motions are thought to be resolved by formation of a DNA loop (Fig-processivity. The replicase also contains a processivity switch that dissociates the polymerase from its ␤ ure 1A) (for overview, see Kornberg and Baker, 1992). As the DNA is extended, the loop grows until the poly-clamp. The switch requires the subunit of the clamp loader and is regulated by different DNA structures. merase bumps into the RNA primer of the fragment it made previously (Figure 1B). At this point, the polymer-At a primed site, the switch is "off." When the replicase reaches the downstream primer to form a nick, the ase must dissociate from the DNA in order that it may start extension of the next Okazaki fragment. Given the switch is flipped, and ejects the polymerase from ␤. This switch has high fidelity for completed synthesis, very low copy number of Pol III holoenzyme (10-20 copies/cell) and the need for 2000-4000 Okazaki fragments, remaining "off" until just prior to incorporation of the last nucleotide and turning "on" only after addition of this lagging-strand polymerase must be rapidly recycled. the last dNTP. These actions of are confined to its C-terminal region, which is located outside the clamp Pol III holoenzyme rapidly cycles to new primed sites despite being held to DNA by a protein ring (O'Donnell, loading apparatus. Thus, this highly processive replication machine has evolved a mechanism to specifi-1987; Stukenberg et al., 1994). Our earlier studies have shown that the polymerase is highly processive during cally counteract processivity at a defined time in the lagging-strand cycle. chain extension, but upon completing DNA, it rapidly disengages from its ␤ clamp and the DNA; the orphaned ␤ clamp is left behind on the duplex product (as illus-Introduction trated in Figure 1B). As synthesis proceeds, the clamp loader can catalytically load another ␤ clamp on a new The replicative DNA polymerase of E. coli, DNA polymerase III holoenzyme (Pol III holoenzyme), is rapid and RNA primed site, leaving it there while the polymerase finishes a fragment. The lagging-strand polymerase, highly processive (Kelman and O'Donnell, 1995). Its processivity, in excess of 50 kb, derives from the ring-upon completing a fragment, dissociates from ␤ and rapidly hops to the new ␤ clamp (Figure 1B). shaped ␤ sliding clamp that encircles DNA and tethers the polymerase to DNA during synthesis (Kong et al., This report outlines the mechanism of this "processivity switch." The results demonstrate that the subunit 1992; Stukenberg et al., 1991). The ␤ clamp is a homodimer of crescent shaped monomer units, and the ring of the clamp loader is required for the processivity switch and that the switch proceeds with extremely high is alternately opened and closed at one interface for assembly onto DNA by the ␥ complex clamp loader fidelity. The polymerase retains high affinity to its clamp even when only one nucleotide remains to be incorpo-(␥ 2 ␦␦Ј) within the holoenzyme structure (Turner et al., 1999). Pol III holoenzyme contains two molecules of DNA rated. Upon closing the gap to a nick, the switch is turned "on," and separates the polymerase from ␤ with polymerase III core (␣, DNA polymerase; ⑀, 3Ј-5Ј exonuclease;) for simultaneous synthesis of both strands of a half-life of about 1 s. The processivity switch requires only the C-terminal domain of , which retains DNA bind-the DNA helix. These core polymerases are crosslinked to the single clamp loader via the two subunits ing activity and is positioned outside the clamp loader apparatus. (McHenry, 1982; Studwell-Vaughan and O'Donnell, 1991; Jeruzalmi et al., 2002). The ␥ and subunits are encoded by the same dnaX gene; (71 kDa) is the full-length Results product, and ␥ (47 kDa) is truncated by a translational frameshift. The 24 kDa C-terminal sequence of binds The Processivity Switch Requires Synthesis both the core and DnaB helicase (see Figure 1A) (Kim to the Very Last Nucleotide et al., 1996; Studwell-Vaughan and O'Donnell, 1991; Gao Our previous studies utilized large circular bacterioand McHenry, 2001a, 2001b). phage templates to demonstrate that Pol III holoenzyme Continuous synthesis of the leading strand at a repliremains tightly bound to ␤ during synthesis, but upon cation fork fits nicely with the picture of a highly progoing full circle and bumping into the 5Ј end of the initiating primer, the polymerase pops off the ␤ clamp (

eLife, Jan 14, 2015

We have reconstituted a eukaryotic leading/lagging strand replisome comprising 31 distinct polype... more 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, Langston 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.

DNA repair, Jan 9, 2015

Processivity clamps that hold DNA polymerases to DNA for processivity were the first proteins kno... more 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.

Proceedings of the National Academy of Sciences, 2008

DNA polymerases attach to the DNA sliding clamp through a common overlapping binding site. We ide... more DNA polymerases attach to the DNA sliding clamp through a common overlapping binding site. We identify a small-molecule compound that binds the protein-binding site in the Escherichia coli β-clamp and differentially affects the activity of DNA polymerases II, III, and IV. To understand the molecular basis of this discrimination, the cocrystal structure of the chemical inhibitor is solved in complex with β and is compared with the structures of Pol II, Pol III, and Pol IV peptides bound to β. The analysis reveals that the small molecule localizes in a region of the clamp to which the DNA polymerases attach in different ways. The results suggest that the small molecule may be useful in the future to probe polymerase function with β, and that the β-clamp may represent an antibiotic target.

Molecular Cell, 2005

This report demonstrates that the ␤ sliding clamp of E. coli binds two different DNA polymerases ... more This report demonstrates that the ␤ sliding clamp of E. coli binds two different DNA polymerases at the same time. One is the high-fidelity Pol III chromosomal replicase and the other is Pol IV, a low-fidelity lesion bypass Y family polymerase. Further, polymerase switching on the primed template junction is regulated in a fashion that limits the action of the lowfidelity Pol IV. Under conditions that cause Pol III to stall on DNA, Pol IV takes control of the primed template. After the stall is relieved, Pol III rapidly regains control of the primed template junction from Pol IV and retains it while it is moving, becoming resistant to further Pol IV takeover events. These polymerase dynamics within the ␤ toolbelt complex restrict the action of the error-prone Pol IV to only the area on DNA where it is required.

Journal of Biological Chemistry, 2003

Replication factor C (RFC) is a heteropentameric AAA؉ protein clamp loader of the proliferating c... more Replication factor C (RFC) is a heteropentameric AAA؉ protein clamp loader of the proliferating cell nuclear antigen (PCNA) processivity factor. The prokaryotic homologue, ␥ complex, is also a heteropentamer, and structural studies show the subunits are arranged in a circle. In this report, Saccharomyces cerevisiae RFC protomers are examined for their interaction with each other and PCNA. The data lead to a model of subunit order around the circle. A characteristic of AAA؉ oligomers is the use of bipartite ATP sites in which one subunit supplies a catalytic arginine residue for hydrolysis of ATP bound to the neighboring subunit. We find that the RFC(3/4) complex is a DNA-dependent ATPase, and we use this activity to determine that RFC3 supplies a catalytic arginine to the ATP site of RFC4. This information, combined with the subunit arrangement, defines the composition of the remaining ATP sites. Furthermore, the RFC(2/3) and RFC(3/4) subassemblies bind stably to PCNA, yet neither RFC2 nor RFC4 bind tightly to PCNA, indicating that RFC3 forms a strong contact point to PCNA. The RFC1 subunit also binds PCNA tightly, and we identify two hydrophobic residues in RFC1 that are important for this interaction. Therefore, at least two subunits in RFC make strong contacts with PCNA, unlike the Escherichia coli ␥ complex in which only one subunit makes strong contact with the ␤ clamp. Multiple strong contact points to PCNA may reflect the extra demands of loading the PCNA trimeric ring onto DNA compared with the dimeric ␤ ring.

Biochemistry, 1997

The disordered N-(1-73) and C-(74-108) fragments of oxidized Escherichia coli thioredoxin (Trx) r... more The disordered N-(1-73) and C-(74-108) fragments of oxidized Escherichia coli thioredoxin (Trx) reconstitute the native structure upon association [Tasayco, M. L., & Chao, K. (1995) Proteins: Struct., Funct., Genet. 22, 41-44]. Kinetic measurements of the formation of the complex (1-73/74-108) at 20°C under apparent pseudo-first-order conditions using stopped-flow far-UV CD and fluorescence spectroscopies indicate association coupled to folding, an apparent rate constant of association [k on) (1330 (54) M-1 s-1 ], and two apparent unimolecular rate constants [k 1) (0.037 (0.007) s-1 and k 2) (0.0020 (0.0005) s-1 ]. The refolding kinetics of the GuHCl denatured Trx shows the same two slowest rate constants. An excess of N-over C-fragment decreases the k on , and the slowest phase disappears when a P76A variant is used. Stopped-flow fluorescence measurements at 20°C indicate a GuHCldependent biphasic dissociation/unfolding process of the complex, where the slowest phase corresponds to 90% of the total. Their rate constants, extrapolated to zero denaturant, k-1) (9 (3) × 10-5 s-1 and k-2) (3.4 (1.2) × 10-5 s-1 , show m # values of (4.0 (0.4) kcal mol-1 M-1 and (3.5 (0.1) kcal mol-1 M-1 , respectively. Our results indicate that: (i) a compact intermediate with trans P76 and defined tertiary structure seems to participate in both the folding and unfolding processes; (ii) not all the N-fragment is competent to associate with the C-fragment; (iii) conversion to an association competent form occurs apparently on the time scale of P76 isomerization; and (iv) the P76A variation does not alter the association competency of the C-fragment, but it permits its association with "noncompetent" forms of the N-fragment.