Initiating & Controlling Dna Replication

Biochemistry 201

Biological Regulatory Mechanisms: Lecture 3

January 14, 2013

INITIATING & CONTROLLING DNA REPLICATION

DISCUSSION

*** Fu, Y.V., Yardimci, H., Long, D.T., Ho, T.V., guainazzi, A., Bermudez, V.P.,Hurwitz, J., van Oijen, A., Scharer, O.D., Walter, J.C. (2011) Selective bypass of a lagging strand roadblock by the eukaryotic replicative DNA helicase. Cell. 146: 931-41.

REVIEWS

1) Gilbert, D.M. (2004). In search of the holy replicator. Nature Rev. Mol. Cell. Biol. 5:848-55

Review of literature on eukaryotic replication origins addressing puzzle of why they are so hard to define in metazoans and suggesting that replicator paradigm that worked for E. coli, S. cerevisiae, and viruses needs to be relaxed. Maybe metazoan origins are determined epigenetically by some level of chromatin structure.

2) Kaguni. (2006). DnaA: Controlling the Initiation of Bacterial DNA Replication and More. Annu. Rev. Microbiol. 60:351-.71.

Most recent description of the mechanisms that contribute to the block to re-initiation of oriC replication. Not the clearest review because opposing papers are presented with little attempt to resolve differences, but does show that there are still a lot of questions of exactly how oriC replication control works, and a field ripe for a Bioreg proposal.

3)Duderstadt, K.E., and Berger, J.M. (2008). AAA+ ATpases in the Initiation of DNA Replication. Crit. Rev. Bioch. Molec. Biol. 43:163-187.

Many of the elongation and initiation proteins involved in replication are part of a large family of ATP binding and hydrolyzing proteins called AAA+ proteins (world’s weakest acronym “ATPases associated with a variety of cellular activities”). This review provides a nice overview of the structure and function of AAA+ proteins involved in replication initiation.

IDENTIFYING INITIATION SITES (PHYSICALLY MAPPING ORIGINS)

6) Huberman, J.A. and Riggs, A.D. (1968). On the Mechanism of DNA Replication in Mammalian Chromosomes. J. Mol. Biol. 32, 327.

A nucleotide incorporation assay visualized by fiber autoradioagraphy that demonstrated that multiple initiation sites fire in concert, each leading to bidirectional replication. Elongation rates and interorigin distances were also measured. Formally this was not a mapping of initiation sites, because there were no physical reference points.

7)Danna KJ; Nathans D. (1972). Bidirectional replication of Simian Virus 40 DNA. Proc. Natl. Acad. Sci. 69, 3097-100.

First physical mapping of replication origins in a nucleotide incorporation assay coupled with the first use of restriction enzymes. We take these methods for granted now, but this was Nobel-prize winning work. This method is limited to small genomes where one can account for all the fragments generated by digestion of a genome.

8)Brewer, B.J. and Fangman, W.L. (1987). The localization of replication origins on ARS plasmids

in S. cerevisiae. Cell. 51, 463-71.

Development of the neutral-neutral gel system for identifying DNA fragments containing origins.

Based on idea that these fragments will contain replication bubbles while they are replicating and that this unique structure confers a distinct migration position on this gel system. In subsequent papers they later modified this technique so that they can determine the direction of fork movement through fragments that do not contain origins (see Brewer, B.J. and Fangman, W.L. Science (1993) 262, 1728-31)

9) Raghuraman, M.K., Winzeler, E.A., Collingwood, D., Hunt, S., Wodicka, L., Conway, A., Lockhart, D.J., Davis, R.W., Brewer, B.J., and Fangman, W.L. (2001). Replication dynamics of the yeast genome. Science. 294:115-121.

Replication entering the brave new world of microarrays. Average replication timing for every chromosomal segment is monitored by DNA copy number and plotted against chromosomal position to obtain a replication profile. Peaks indicates origins, valleys termination sites, peak height indicates either origin timing and/or firing efficiency, peak slopes indicate elongation rates.

IDENTIFYING REPLICATOR ELEMENTS (GENETICALLY MAPPING ORIGINS)

10)Oka, A., Sugimoto, K., Takanami, M., and Hirota, Y. (1980). Replication origin of the Escherichia

coli K-12 chromosome: the size and structure of the minimum DNA segment carrying the information for autonomous replication. Mol. Gen. Genetics. 178, 9-20.

Cloning of oriC by shotgun method into colE1 plasmid and selecting ability of the plasmid to be maintained because it contains a replicator.

11) Marahrens, Y. and Stillman, B. (1992). A Yeast Chromosomal Origin of DNA Replication Defined by Multiple Functional Elements. Science. 255, 817-823.

Use of linker scanning analysis to dissect the structure of the B domain of an ARS. Previous analyses using simple deletion analysis had resulted in very ambiguous conclusions about this domain and the lack of sequence conservation had discouraged attempts to use point mutagenesis.

12) Aladjem, M.I., Rodewald, L.W., Kolman, J.L., and Wahl, G.M. (1998). Genetic dissection of a mammalian replicator in the human beta-globin locus. Science. 281, 1005-1009.

An example of mapping initiation sites by PCR analysis of newly synthesized daughter strands. More importantly, this paper combines genetic and physical origin mapping techniques to argue that specific origin sequences actually exist in mammalian cells (a subject of debate and contention). The genetic strategy for identifying origins has classically used autonomous plasmid replication as the assay for origin function, but this strategy has failed dismally in mammalian systems. The authors overcome this problem by integrating their sequences at specific loci in the mammalian genome using site specific recombination and using PCR analysis to show that these sequences induce new initiation sites at the integration loci. Unfortunately, further attempts to refine the sequence analysis has proven difficult consistent with other experiments suggesting that initiation may at best be confined to broad zones of tens of kilobases.

34) Dershowitz, A. and Newlon, C.S. (1993). The Effect on Chromosome Stability of Deleting Replication Origins. Mol. Cell. Biol. 13, 391.

Latest in a series of papers that examine the effects of deleting replication origins from yeast chromosome III. Deleting single origin has no demonstrable effect showing a considerable redundancy of origins. You have to delete many origins before you see high rate of chromosome loss. Thus, yeast origins are highly redundant.

INITIATION OF E. COLI DNA REPLICATION

13)Fuller, R.S., Kaguni, J.M. and Kornberg, A. (1981). Enzymatic replication of the origin of the E.

coli chromosome. Proc. Natl. Acad. Sci. USA 78, 7370-7374.

Breakthrough in establishing conditions in vitro for replication initiation at the E. coli origin. No

substitute for blood, sweat, and tears (lots of tears) in setting up great in vitro systems.

14) van der Ende, A, Baker, T.A., Ogawa, T., and Kornberg, A. (1985). Initiation enzymatic replication at the origin of the Escherichia coli chromosome: Primase as the sole priming enzyme. Proc. Natl. Acad. Sci. USA 82, 3954.

Initial dissection of the initiation reaction, starting with the description of a presynthesis step, a step during which no nucleotide incorporation is occurring but something is happening that allows later incorporation to occur. Uses kinetics, dNTP depletion, and temperature shifts to perturb the reactions and identify intermediates that form during initiation.

15)Baker, T.A., Sekimizu, K., Funnell, B.E., and Kornberg, A. (1987). Extensive Unwinding of the Plasmid Template during Staged Enzymatic Initiation of DNA Replication from the Origin of the Escherichia coli Chromosome. Cell. 45, 53-64.

Excellent use of serendipity (or the "prepared mind") in the development of an unwinding assay to

define more precisely what DnaA, DnaB, DnaC and SSB does to the DNA during presynthesis. These proteins cause the DNA to be highly underwound and to migrate faster than standard supercoiled DNA on a gel.

16)Funnell, B.E., Baker, T., and Kornberg, A. (1987). In vitro Assembly of a Prepriming Complex at the Origin of the Escherichia coli Chromosome. J. Biol. Chem. 262, 10327-10334.

Direct examination of DnaA and DnaB (and not DnaC) protein on the origin by immunoEM.

17)Baker, T.A., Funnell, B.E., and Kornberg, A. (1987). Helicase Action of DnaB Protein During

Replication from the E. coli Chromosomal Origin in vitro. J. Biol. Chem. 262, 6877.

Complex loading and bidirectional unwinding (with DnaB at the ends of the bubbles) seen directly by EM.

*18)Bramhill, D. and Kornberg, A. (1988). Duplex Opening by DnaA Protein at Novel Sequences in

Initiation of Replication at the Origin of the E. coli Chromosome. Cell 52, 743.

Shows that DnaA protein bound to ATP is sufficient to open the DNA duplex at oriC. SS vs DS specific probes are used to monitor the unwinding at oriC and to demonstrate that the duplex is opened at the 13 bp AT-rich repeats.

19) Speck, C. and Messer, W. (2001). Mechanism of origin unwinding: sequential binding of DnaA to double- and single-stranded DNA. EMBO J. 20, 1469-76.

Systematic analysis of DnaA-ATP binding to double and single-stranded motifs in oriC leads to a proposal for how DnaA-ATP can unwind oriC using just binding energy.

20)Fang, L., Davey, M.J., and O’Donnell, M. (1999). Replisome Assembly at oriC, the Replication Origin of E coli, Reveals an Explanation for Initiation Sites outside an Origin. Mol. Cell 4, 541-533.

Targeted mutagenesis of ATP hydrolytic activity of DnaB helicase allows O’Donnell’s group to examine the loading of DnaB without having to worry about its helicase activity. Protection assays on the unwound single-stranded DNA shows that two DnaB helicase complexes are initially positioned in a manner that requires them to move past each other to get to their respective forks.

*21)Davey, M.J., Fang, L., McInerney, P., Georgescu, R.E., and O’Donnell, M. (2002). The DnaC helicase loader is a dual ATP/ADP switch protein. EMBO J. 21, 3148-59.

Careful analysis of role of ATP binding and hydrolysis by DnaC in the binding, loading, and inhibition of DnaB. This is a beautiful example of how the power of biochemistry in dissecting a mechanisms can be greatly enhanced by the use of targeted genetics. Key to this paper was having special mutants in the ATP binding pockets of DnaB and DnaC, allowing the role of these pockets to be distinguished. The hydrolysis of DnaC acts as a switch, but it is not simply switching between an active and inactive form. The ATP bound form is active for early functions in the loading cycle, but inhibitory for later functions. The ADP bound form is inactive for the early functions, but permissive for the later functions. The hydrolysis ensures that both forms act in the proper order to execute the loading of DnaB.

CONTROL OF ORIC INITIATION DURING THE E. COLI CELL CYCLE

22)Helmstetter, C.E. and A.C. Leonard (1987). Coordinate Initiation of Chromosome and Minichromosome Replication in E. coli. J. Bacteriol. 169, 3489.

Demonstration that small plasmids that replicate using oriC fire simultaneously with the chromosome origin. Shows that "initiation potential" is restricted to brief windows of time in the bacterial cell cycle.

23) Russell, D.W., and N.D. Zinder (1987). Hemimethylation prevents DNA replication in E. coli. Cell 50, 1071-1079.

Indirect evidence that hemimethylated oriC DNA is refractory to initiation. Key was synthesizing and presenting templates of defined methylation state to cells via transformation. Transformation frequency is only an indirect assay for replication, so the title is a bit overstated, but the hypothesis they generated was a good working model that proved true.

24)Boye, E. and Lobner-Olesen, A. (1990). The Role of dam methlytransferase in the control of DNA replication in E. coli. Cell 62, 981.

Demonstration that if the level of dam methylase in the cell is too high or too low, initiation of replication at the multiple oriCs in a cell occurs asynchronously. In other words the number of oriCs in a cell deviates from the expected numbers of 1, 2, 4, 8, or 16. Copy number of oriC is counted by simultaneously inhibiting replication initiation and cell division, letting the replication forks complete replication, and counting the total number of genomes by flow cytometry. Cells with 3, 5, 6, or 7 genomes are scored in an asynchrony index. This is a very indirect assay for uncontrolled initiation, but was the first genetic evidence that dam methylase might contribute to the control of replication initiation.

*25) Campbell, J.L. and Kleckner, N. (1990). E. coli oriC and the DnaA gene promoter are sequestered from the dam methyltransferase following the passage of the chromosomal replication fork. Cell 62, 967.

Experimental demonstration using synchronized cell cultures that oriC appears to be protected from remethylation soon after replication initiation. Key is using methylation sensitive restriction enzyme to monitor hemimethylation state of one of the oriC GATC sites. Led to the idea that hemimethlyated oriC is sequestered from dam methylase and that a similar sequestration from initiation proteins could help prevent immediate re-initiation. What that sequestration ex

26)Landoulsi, A., Malki, A., Kern, R., Kohiyama, M., and Hughes, P. (1990). The E. coli cell surface specifically prevents the initiation of DNA replication at oriC on hemimethylated DNA templates. Cell. 63, 1053-1060.

E. coli outer membrane preps, which were first shown by Schaecter’s group to bind specifically to hemimethylated oriC, are now shown to specifically inhibit in vitro replication of hemimethylated oriC templates. Inhibition appears to be through preventing DnaA binding to oriC. A potential assay for purifying the putative sequestration activity, but the biochemists were beat out by the geneticists (see Lu et al. #29).

27)Lu, M., Campbell, J.L., Boye, E., and Kleckner, N. (1994). SeqA: a negative modulator of replication initiation in E. coli. Cell. 77, 413-426.

Kleckner's group hypothesizes that the failure of hemimethylated DNA to replicate is due to active inhibition of oriC rather than a failure to activate oriC. They devise a genetic screen for loss of function mutations that allow methylated DNA to transform dam(-) strains efficiently, using the Russell and Zinder presumption that the failure to transform is due to a failure to replicate. This leads to seqA. Mutation impairs sequestration of hemimethylated oriC and disrupts initiation synchrony, but cells are viable and the most significant effect observed is asynchrony of initiation. Even later direct analysis of DNA content by flow cytometry (PNAS 1996. 93:12206-211), showed only a slight propensity to over-replicate. Clearly not the entire story.

28) Nievera, C., Torgue, J.J., Grimwalde, J.E., and Leonard, A.C. (2006). SeqA blocking of DnaA-oriC interactions ensures staged assembly of the E. coli pre-RC. Mol. Cell. 24:581-92.

Translating the general concept of "sequestration" to a specific biochemical mechanism has been difficult and controversial. In this paper the authors monitor DnaA binding in vivo by genomic footprinting. They argue that SeqA allows DnaA to bind its high affinity sites on the hemimethylated oriC but prevents DnaA from binding its low affinity sites where origin unwinding occurs.

*29)Sekimizu, K., Bramhill, D., and Kornberg, A. (1987). ATP activates DnaA protein in initiating replication of plasmids bearing the origin of the E. coli chromosome. Cell. 50, 259-265

Discovery that ATP has an allosteric role in activating DnaA for initiation. Moreover, the ADP bound form is inactive for initiation and the two forms can interconvert by slow hydrolysis and slow nucleotide exchange. Raises the possibility of a regulatory role for ATP/ADP in restricting DnaA function in the cell to a brief window of the cell cycle (see ref 24).

30) Kurokawa, K., Nishida, S., Emoto, A., Sekimizu, K., and Katayama, T. (1999). Replication cycle-coordinated change of the adenine nucleotide-bound forms of DnaA protein in Escherchia coli. EMBO J. 18, 6642-52.

Nucleotide bound state of DnaA in vivo fluctuates in synchronized cell populations, consistent with this state helping to regulate DnaA initiation potential. Extremely tight binding affinity of ATP and ADP for DnaA allowed IP of DnaA and analysis of bound nucleotide. Regeneration of the ATP bound "active" state of DnaA requires new protein synthesis consistent with an old observation that each new round of initiation requires new protein synthesis. Suggests that synthesis of new DnaA (which would bind ATP, the most abundant nucleotide in the cell) is the primary means of regenerating active DnaA. However, in other papers Kornberg's lab explored the stimulation of DnaA nucleotide exchange by phospholipids suggesting a second potential pathway for regenerating active DnaA that involves membrane association. Regeneration of DnaA-ATP is still an open question.

*31) Katayama, T., Kubota, T., Kurokawa, K. Crooke, E., and Sekimizu, K. (1998). The initiator function of DnaA protein is negatively regulated by the sliding clamp of the E. coli chromosomal replicase. Cell. 94, 61-71.

Intrinsic ATP hydrolysis of DnaA in the allosteric ATP binding site is very slow, but can be greatly stimulated by an activity detected in crude extracts. When this activity is purified it separates into 3 factors, one of which is purified and turns out to be the beta subunit (clamp). The other two requirements are an unpurified factor called IdaB and clamp loading activity (provided by gamma complex or PolIII* holoenzyme). For the beta subunit to stimulate DnaA's ATP hydrolysis it must be loaded onto DNA by the clamp loader. Provides a potential means of coupling initiation of DNA synthesis to inactivation of DnaA for re-initiation.

32) Kato, J. and Katayama, T. (2001). Hda, a novel DnaA-related protein, regulates the replication cycle

in Escherichia coli. EMBO J. 20, 4253-62.

Further purification of IdaB activity was very difficult, and it was genetics that led to its identification. Taking a page from the eukaryotic replication field, the authors looked for new replication mutations by looking for elevated plasmid loss. One of these mutants (in PCNA) was suppressed by high copy Hda. Hda is detectable in the IdaB fractions and large quantities of purified Hda can substitute for IdaB in the biochemical assay that inactives DnaA-ATP . Conditional (ts) mutants of Hda show subtle signs of over-initiation when shifted to restrictive temperatures. Consistent with Hda having some but not the entire responsibility of preventing re-initiation. No one has yet combined the hda and seqA mutants in a conditional manner.

33) Nishida, S., Fujimitsu, K., Sekimizu, K., Ohmura, T., Ueda, T., and Katayama, T. (2002). Negative control of DNA replication by hydrolysis of ATP bound to DnaA protein, the initiator of chromosomal DNA replication in Escherichi coli. J. Biol. Chem. 277, 14986-95.

Targeted mutagenesis of the ATP binding pocket of DnaA generates a mutant with normal ATP binding and initiation activity but defective ATP hydrolysis in vitro and in vivo (authors are lucky since this is a difficult mutation to obtain). Overexpression of this mutant protein induces more re-initiation than overexpression of wild-type DnaA, suggesting a role for ATP hydrolysis in preventing re-initiation.

SOME QUESTIONS TO THINK ABOUT THE LECTURE

1) Could you use electron microsopy to determine the site of initiation? Would it work for small genomes or large? What would you use as a physical reference point? Could you distinguish bidirectional replication from unidirectional replication?

2) Can you map the site of replication initiation by incorporation of radioactive precursors. How would you do it for small genomes (like a eukaryotic virus under 50 kb). How would you do it for a cellular genome?