Eukaryotic DNA replication is initiated in a tightly regulated manner. In the first step the pre-replicative complex (pre-RC) forms by the step-wise assembly of origin-recognition complex (ORC), Cdc6, Cdt1 and MCM2-7 complex onto chromatin [119]. Electron mi-croscopic studies with purified yeast MCM2-7 showed that the loaded complex encircled
1.3. Eukaryotic DNA replication 9 double-stranded DNA after it was loaded onto origins [120]. In the second step the pre-RC is activated to a replisome; its activation depends on the activities of the S- phase kinases Dbf7-Cdc7 (DDK) and CDK [121]. The loading of Cdc45 and the GINS complex (a het-erotetramer of Sld5, Psf3, Psf2 and Psf1) onto MCM2-7 requires multiple protein factors which minimally include Sld2, Sld3, Dbp11, MCM10, Ctf4 and DNA polymerase (pol)!in yeast [122]. The homologues in higher eukaryotes are presumably ReqQL4 (Sld2), Treslin (Sld3) and TopBP1 (Dbp11). Treslin is essential for cell viability and has been identi-fied through its interaction with TopBP1 in Xenopus [123]. In budding yeast, Sld2 and Sld3 are the only factors involved in the loading process that need to be phosphorylated [122, 124].
The complex of Cdc45, GINS and MCM2-7 (CMG) constitutes the replicative helicase (fig. 1.4) that unwinds origins and contributes to the recruitment of replicative poly-merases to the replisome. In vivo experiments in Hela cells showed that the interactions between the CMG complex members only occur at the G1/S transition of the cell cycle and required CDK and DDK activities [125]. The formation of CMG also required a number of the factors that were essential for formation of the yeast replisome progression complex (RPC), such as Ctf4 and TopBP1. The omission or selected degradation of any of the CMG components of the yeast replisome progression complex abolished replication fork movement and displaced other members of the RPC [126, 127]. Also, in the Xenopus cell free system the CMG subunits interacted at stalled replication forks, indicating the presence of a stable complex in this organism [128]. Finally, Botchan’s group isolated the CMG complex fromDrosophila melanogaster (dm) embryos and further characterized the dm CMG complex isolated from the Sf9/Baculovirus system [129, 130]. In vitro the dm CMG complex unwinds DNA in a 3’ → 5’ orientation and thus moves on the leading strand.
Fig. 1.4: Model of the human replisome. The complex of Mcm2-7, Cdc45 and GINS, the CMG, unwinds the duplex DNA ahead of leading and lagging strand polymerase, assigned as Pol$and Polδ, respectively. PCNA increases the processivity of the polymerases. Ctf4 and Mcm10 are representatives of additional factors at the replication fork. Adapted from [131].
The third manuscript enclosed in this thesis describes the isolation and characterization of the human (h) CMG complex from the Sf9/Baculovirus system. As shown with the dm CMG, the h CMG complex requires ATP, magnesium and ss DNA for its loading on
DNA. Forked DNA structures and dT-rich sequences enhance the 3’→5’ helicase activity of the h CMG complex. Single-strand binding protein from E. coli, as well as its human homolog, hRPA, stimulated the helicase processivity.
The MCM2-7 complex provides the motor of the replicative helicase. The laboratory of Anthony Schwacha showed that yeast MCM2-7 complex possessed in vitro helicase activity using high ionic strength conditions [132]. This helicase activity, however, has not yet been reproduced to date for complexes isolated from higher eukaryotes. The association with Cdc45 and GINS, however, activates the helicase activity ofDrosophila melanogaster MCM2-7 [130]. Since Cdc45 and GINS lack enzymatic activities it is assumed that they activate the helicase activity and participate with the other replicative proteins at the fork.
Previous studies in our laboratory showed that the GINS complex weakly interacts with all three replicative polymerases and stimulates pol! and pol αand to a lesser extent pol δ [133]. This indicates a physical link between the replicative helicase and polymerases.
In yeast, pol ! was shown to associate with replication forks during S phase [134]. It is essential in budding and fission yeast [135, 136]. In human cells, in vivo studies showed that pol ! depletion leads to a more dramatic effect than the depletion of polδ [133].
Human pol ! is a heterotetramer consisting of p261/Pol2, p59, p17 and p12 subunits.
The N-terminus of the p261 subunit is solely responsible for DNA polymerase activity.
In in vitro primer extension assays using the single-stranded DNA plasmid M13mp18, pol ! showed a higher processivity of DNA synthesis than pol δ. Pol δ was originally identified from calf thymus [137] and further characterized using the SV40 replication system (see below) [138]. It is a heterotetramer composed of p124, p66, p51 and p12 (reviewed in [139]). In budding yeast, in vivo studies indicated that pol ! duplicates leading strand while pol δ synthesizes lagging strands (fig. 1.4). Point mutations in the endogenous polymerases lead to distinct misincorporations in the replicated DNA and the travel pattern from known origins of replication was traced by genome-wide analysis using the misincorporations as travel markers [140, 141]. The selective action of these polymerases, pol ! and pol δ, in vitro remains to be established.
The in vitro system using the simian virus (SV) 40 origin DNA with the protein SV40 T Antigen helped identify proteins of the eukaryotic replication fork (reviewed in [142–
145]). In the SV40 replication system, T Antigen assembles as a hexamer at SV40 ori+ and unwinds the DNA [146–149]. Thus, T Antigen alone replaces the activities due to the combined action of the bacterial DnaA, DnaC and DnaB as well as the human ORC, Cdc6, Cdt1 and MCM2-7 proteins. Human RPA then binds to the single stranded DNA [150–153]. And although E. coli SSB can substitute for hRPA in DNA binding, hRPA is required for the interaction of T Antigen with human polα/primase complex [151, 154–
157]. The recruited primase catalyzes the synthesis of primers that pol α then extends to fragment lengths of about 40 to 50 nucleotides [158–160]. The clamp loader RFC competes with polα/primase at the primer-template junction and displaces pol α. RFC then loads the clamp PCNA in an ATP-dependent manner and thereby recruits pol δ to the DNA [159, 161, 162]. This system allowed the initial characterization of a number of replication proteins. However, it does not involve the human replicative helicase, as T
1.3. Eukaryotic DNA replication 11 Antigen serves as both the origin binding protein and helicase. Also, in the SV40 system pol δ replicates both the leading and lagging strand. However, in vivo experiments in Xenopus and human cells showed the requirement for pol ! in DNA replication [133].
In vivo experiments indicate that pol ! crosslinked to human DNA, while it was not crosslinked to SV40 DNA [163].
In the third manuscript initial efforts were undertaken to remodel the human replication fork. Using a primed minicircle, we showed that the coupling of the hCMG with human pol!, but not pol δ, formed fragments that are larger than 10 kilobases. The production of these large fragments is in keeping with the action of leading strand polymerization.
The results also suggest that pol ! may directly contact the CMG complex and increase its processivity.
Chapter 2
A novel DNA nuclease is stimulated by association with the GINS
complex
Zhuo Li,1,2 Miao Pan,1,2 Thomas J. Santangelo,3 Wiebke Chemnitz,4 Wei Yuan,1,5 James L. Edwards,1,5 Jerard Hurwitz,4 John N. Reeve,3 and Zvi Kelman1,2,∗
Nucleic Acids Res. 2011 August; 39(14): 6114–6123.
1Institute for Bioscience and Biotechnology Research, 9600 Gudelsky Drive, Rockville, MD 20850
2Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD 20742
3Department of Microbiology, Ohio State University, Columbus, OH 43210
4Program in Molecular Biology, Memorial Sloan Kettering Cancer Center, New York, NY 10065
5Department of Chemistry and Biochemistry, University of Maryland, College Park, MD 20742, USA
*To whom correspondence should be addressed.
13
2.1 Abstract
Chromosomal DNA replication requires the spatial and temporal coordination of the activities of several complexes that constitute the replisome. A previously uncharac-terized protein, encoded by TK1252 in the archaeon Thermococcus kodakaraensis, was shown to stably interact with the archaeal GINS complex in vivo, a central component of the archaeal replisome. Here, we document that this protein (TK1252p) is a proces-sive, single-strand DNA-specific exonuclease that degrades DNA in the 5’→3’ direction.
TK1252p binds specifically to the GINS15 subunit ofT. kodakaraensisGINS complex and this interaction stimulates the exonuclease activity in vitro. This novel archaeal nucle-ase, designated GINS-associated nuclease (GAN), also forms a complex in vivo with the euryarchaeal-specific DNA polymerase D. Roles for GAN in replisome assembly and DNA replication are discussed.