DNA

Replica,on

1. Topics covered in this module

Semi-Conserva,ve DNA Replica,on Overall Principles of DNA Replica,on Telomere Replica,on Mitochondrial DNA replica,on

2. Why does this topic matter?

Stem Cells (Stem cells are powerful tools in biology and medicine. What can scien,sts do with these cells and their incredible poten,al?) Cancer (Can cuHng-edge research lead to preven,on and treatment strategies that could make cancer obsolete?) Synthe,c Biology (Making Life from Bits and Pieces)

 In their paper in Nature announcing the discovery of the double helix structure of DNA, Watson and Crick (1953) made one of the most famous statements in molecular biology:

It has not escaped our no,ce that the specic pairing we have postulated immediately suggests a possible copying mechanism for the gene,c material.

DNA replica,on research has been driven by three related but dis,nct issues:
The topological problem Early 1980s: DNA topoisomerases. The replica0on process During the 1960s: replica,on steps in E. coli, followed by details of eukaryo,c genome replica,on. This work is ongoing, with research centered on topics such as the ini,a,on of replica,on and the precise modes of ac,on of the proteins ac,ve at the replica,on fork. The regula0on of genome replica0on, par,cularly in the context of the cell cycle, has become the predominant area of research in recent years. This work has shown that ini,a,on is the key control point in genome replica,on.

Supercoiling

Supercoiling of DNA
Topology A. Right handed supercoiling= nega,ve supercoiling (underwinding) B. Le\ handed supercoiling= posi,ve supercoiling C. Relaxed state is with no bends D. DNA must be constrained: plasmid DNA or by proteins E. Unraveling the DNA at one posi,on changes the superhelicity F. Topology only dened for con,nuous deforma,on -no strand breakage

Supercoiling of DNA

Topoisomerase activity

The topological problem

Topoisomerase I
Cut one strand of double-stranded DNA, relax the strand, and reanneal the strands
IA: can only relax nega,ve supercoiled DNA (prokaryotes) IB: solves the problem of overwound and underwound (also referred to as posi,vely or nega,vely supercoiled) (eukaryotes) IC: (archeae)

Topoisomerase II
Cut both strands of the DNA helix simultaneously, Use the hydrolysis of ATP
IIA: form double-stranded breaks with four-base pair overhangs (span all domains of life) IIB: form double-stranded breaks with two base overhangs (found in archaea and some higher plants)

The Meselson-Stahl experiment (1958)

DNA replica0on is semiconserva0ve

DNA replica0on is semidiscon0nuous

DNA Replica,on Steps

Initiation: involves the assembly of a replica,on fork (bubble) at an origin of replica,on sequence of DNA. The fork is generated by a complex of proteins called a primosome. Elongation: addi,on of bases by another complex of proteins called the replisome. Parental strands unwind and daughter strands are synthesized. Termination: the duplicated chromosomes separate from each other.

Similar func,ons are required at all replica,on forks

Function Helicase Loading helicase/ primase Single strand maintenance Priming Slinding clamp Clamp loader (ATPase) Strand elongation RNA primer removal Ligation of Okazaki fragments E. coli DnaB DnaC SSB DnaG (primase) complex Pol III Pol I Ligase Eukaryotes MCM complex cdc6 RPA Pol / primase PCNA RFC Pol / Pol Fen-1, Rnase H1 Ligase I

Enzymes and reac,ons in the DNA replica,on fork

Zheng L , Shen B J Mol Cell Biol 2011;3:23-30

All DNA polymerases have similar structures

DNA polymerases are enzymes that make a complimentary DNA strand from a DNA/RNA template, adding DNA nucleo,des to the 3'-end of the appropriate primer. Although the amino acid sequences of these enzymes were very dissimilar all share some structural similari,es, including a U-shaped DNA binding cle\ that has "ngers," "thumb," and "palm" subdomains. They all have metal ions (usually magnesium) in their ac,ve site. During nucleo,dyl transfer, these metal ions play a key role in interac,ng with the phosphates of the nucleo,des and the 3'-end of the primer. These metal binding sites are highly conserved among dierent DNA polymerases, which highlights their importance for proper func,on of nucleo,de polymeriza,on.

All DNA polymerases have similar structures

Comparison of the structures of four dierent DNA polymerases in complex with DNA

Structure (1999), vol. 7: pages R31-R35

The eukaryo,c DNA polymerases

1) High fidelity replicases
Polymerase Function Pol (alpha) Pol (epsilon) Pol (delta) Pol (gamma) Priming DNA synthesis during replication and repair DNA replication of leading strand during replication and repair (BER, DSBR, NER) DNA replication of lagging strand during replication and repair (BER, DSBR, MMR, NER) Mitochondrial replication and repair Structure 350 kD tetramer 350 kD tetramer 250 kD tetramer 200 kD dimer

The eukaryo,c DNA polymerases (cont.)

2) High fidelity repair
Polymerase Function Pol (beta) Pol (eta) BER, DSBR Translesion DNA synthesis (thymine dimer bypass, relatively accurate replication) Structure 39 kD monomer monomer

The eukaryo,c DNA polymerases (cont.)

3) Low fidelity (error-prone) repair
Polymerase Pol (zeta) Pol (theta) Pol (iota) Pol (kappa) Function Translesion DNA synthesis (thymine dimer bypass) Repair of DNA interstrand cross-links Translesion DNA synthesis (required during meiosis) Translesion DNA synthesis (deletion and base substitution), DSBR (nonhomologous end joining) DSBR (nonhomologous end joining) DNA cross link repair?? Abasic site synthesis (deoxycytidyl transferase activity inserts C across from a nucleotide lacking a base)

Pol (lambda) Translesion DNA synthesis Pol (mu) Pol (nu) Rev1

The eukaryo,c DNA polymerases (cont.)

Polymerase

Function

Terminal Cell-specic, template independent polymerase that deoxynucleotidyl adds nucleotides nearly randomly to coding ends during transferase (TdT) V(D)J recombination

Animations: 1) hfp://www.wiley.com/college/praf/0471393878/student/ anima,ons/dna_replica,on/index.html 2) hfp://www.dnalc.org/resources/3d/03-mechanism-of- replica,on-basic.html 3) Very complete/complex model hfp://www.dnareplica,on.net/model/model.html 4) Trombone model in prokaryotes hfp://www.courses.fas.harvard.edu/~biotext/anima,ons/ replica,on1

 The replica,on program in higher eukaryotes is under a dynamic and plas,c regula,on within a single cell, or within the cell popula,on, or during development. One of the most striking features of DNA replica,on in higher eukaryotes is its plas,city:
1) A best-known example of plasticity is the very rapid replication of the entire genome of the fertilized eggs of amphibians (within a matter of 2030 min), 2) compared with the 810 h required for the genome replication in somatic cells.

Where, when, and how

The challenges facing our understanding of eukaryo,c DNA replica,on can be summarized by three ques,ons:
1) Where in the genome and within the nuclei does DNA replication take place? 2) When within the S phase does each replication origin start firing? 3) How is the entire process of DNA replication regulated?
Ann. Rev. Biochem. (2010): 79: 89-130

1) Where?
Recogni,on by the ini,ator Origin Recogni,on Complex (ORC; heterohexameric complex ORC1- ORC6, 120; 72; 62; 56; 53; 50 kD)

In the budding yeast, where ORC was first identified, it specifically recognizes a 11-bp or 17-bp AT rich sequence known as autonomously replicating sequences (ARS). However:
In fission yeast ORC recognizes DNA through the AT-hook motif present on the ORC4 subunit and preferentially binds adenine/ thymine stretches In Drosophila ORC also shows some preference for AT-rich sequences the human ORC binds to any DNA without apparent sequence specificity

ARS1 Box A is 15 bp long, and contains an 11 bp consensus sequence: 5- (T/A)TTTA(T/C)(A/G)TTT(T/A) -3

Origin recogni,on complex and minichromosome maintenance complex-binding sites as poten,al origins. The minichromosome maintenance (MCM) complex, a heterohexameric complex of MCM 2-7 (98.5; 107.5; 105; 86.4; 113; 94.9 kDa), is loaded onto the ORC bound to chroma,n at late mitosis (M) to early G1 to generate a pre-Replica,ve Complex (pre-RC).

When?
Timing of DNA Replication within the S phase. In budding yeast, early- and late-ring origins have been clearly iden,ed. In ssion yeast, the deni,on of early- and late-ring origins is less clear. The trans-acting factors determining replication timing. Firing of replication origin depends on the phosphorylation events mediated by two kinases, the cyclin-dependent kinase (CDK) and the Dbf4-dependent Cdc7 kinase (Cdc7-Dbf4 or DDK)

How?
Assembly of the prereplica,ve complex and its regula,on. The processes of replica,on ini,a,on are divided into two steps: 1) In the rst step, the ORC, Cdc6, Cdt1, and MCM2-7 proteins are sequen,ally assembled on the chromosome at the late M to early G1 phase, genera,ng a pre-RC. 2) In the second step, pre-RCs are ac,vated to generate ac,ve replica,on forks. At the G1-S transi,on, the ac,vi,es of two kinases, the Cdc7-Dbf4 and the CDK, facilitate the loading of other essen,al replica,on proteins onto the pre- RC to ac,vate the replica,ve DNA helicase and ini,ate chain elonga,on by DNA polymerases.

 Loading of the MCM complex onto DNA is referred to as DNA replica,on licensing. Eukaryo,c cells can only ini,ate DNA replica,on at a specic point in the cell cycle: the beginning of S phase.

Annu. Rev. Biochem. 2005. 74:283315

hfp://www.dnareplica,on.net/model/model.html Prokaryotes trombone model hfp://www.courses.fas.harvard.edu/~biotext/anima,ons/replica,on1

Telomere maintenance
Replica,on of linear chromosomes presents a special problem. The end replica0on problem: during lagging strand replica,on a primase creates RNA/ DNA hybrids to which DNA polymerase binds and extends to form okazaki fragments RNA primers removed and replaced with DNA by DNA polymerase at 5 end, DNA pol cant bind to ll in 3 overhang For every round of replica0on, the DNA would shorten.

Telomeres
problem solved by a terminal 3 overhang which allows priming outside of useful DNA this overhang is part of the Telomere (tandem repeats of a G rich sequence) telomeres also prevent fusion of chromosome ends through blunt ends (i.e., they cap chromosomes) telomere allows full synthesis of DNA end as it is now primed from overhang

TAR (subtelomeric region)

hfp://www.nature.com/scitable/content/telomeres-and-telomerase-34864

Telomeres (cont.)
Sequence is evolu,onary conserved from yeast to ciliates to plants and mammals Human telomeres: 1.000s repeat TTAGGG
!5'...TTAGGG TTAGGG TTAGGG TTAGGG TTAGGG TTAGGG..3'! !3'...AATCCC AATCCC AATCCC ...... ...... ........5!

Telomerase
Telomerase is an enzyme that adds telomere repeat sequences to the 3' end of DNA strands. By lengthening this strand, DNA polymerase is able to complete the synthesis of the "incomplete ends" of the opposite strand. Ribonucleoprotein: Its single snoRNA molecule called TERC ("TElomere RNA Component") provides an AAUCCC (in mammals) template to guide the inser,on of TTAGGG. Its protein component called TERT ("TElomere Reverse Transcriptase") provides the cataly,c ac,on. Thus telomerase is a reverse transcriptase; synthesizing DNA from an RNA template.

 Although vertebrates, ciliates, and yeast have widely divergent TRs, they all contain a template and a puta,ve pseudoknot. In contrast to the species variability of TR, TERTs are highly conserved among eukaryotes.

 Most normal soma,c cells do not express telomerase and, consequently, telomere length gradually decreases with age in nearly all human ,ssues. However, telomerase is ac,ve in other prolifera,ve ,ssues, as in early embryonic and fetal cells, stem cells, germline cells, inammatory cells, and cells in other periodically or con,nuously renewing ,ssues. Moreover, telomerase is consistently ac,ve in the majority of human cancer cells. In humans, telomerase consists of a 451 nt RNA, a cataly,c protein component (human telomerase reverse transcriptase [hTERT]), and a variety of other proteins. hTERT is a 120 kDa protein that contains a reverse transcriptase domain.

 Muta,ons in the RNA component of hTR that aect telomerase ac,vity have also been linked to inherited human disorders of the haemopoie,c system such as autosomal dominant dyskeratosis congenita (DKC) and aplas,c anemia (AA).

Summary:
Analysis of telomeric DNA showed that it generally consisted of repe,,ve DNA of the form TxAyGz. In Tetrahymena, the sequence is TTGGGG, while in humans the sequence is TTAGGG. In humans, this sequence is repeated for 2-10 kb in double- stranded form and then ends in ~100-200 nts of a single-stranded extension of T2AG3; aka, the G-strand overhang. In several species, it appears as if the G-strand can fold back and invade the ds T2AG3 por,on, crea,ng a triplex t-loop structure. This is presumed to sequester the end from recombina,on/repair reac,ons within the cell. Biochemical, molecular and gene,c characteriza,on demonstrated that telomerase (hTERT in humans) is a mul,subunit, ribonucleoprotein complex with reverse transcriptase ac,vity.

Secondary structure of human telomerase RNA (451 nts)

Mol. Cell (2005) 17:671-682

Nt conserva,on is indicated by green lefers and bars. Red lefers and bars indicate the sites of disease-related muta,ons and dele,ons.

Mol. Cell (2005) 17:671-682

Mol. Cell (2005) 17:671-682

In yeast

Synthesis of telomeric DNA by human telomerase

Telomere anima,on hfp://spine.rutgers.edu/cellbio/assets/ash/tel.htm

Telomerase: aging, cancer, and disease

Most soma,c cells have low or undetectable level of telomerase ac,vity. Telomere length is correlated with cellular aging. Telomerase is ac,ve in some germline, epithelial, and haemopoei,c cells, and in >90% of cancer cell lines. Muta,ons in the RNA component of human telomerase have been linked to autosomal dominant dyskeratosis congenita and some forms of aplas,c anemia. Telomerase is required for telomere maintenance. Telomerase is responsible for the immortal phenotype of cancer cells.

The role of telomerase deciency in mammalian aging

Mice whose genes for telomerase have been "knocked out" (either Tert/ or Terc / show many of the degenera0ve changes associated with aging: The number of mitochondria in their cells decreases, as does the func,on of those that remain. Oxygen consump,on and ATP produc,on declines. The eciency of the electron transport chain decreases. This leads to an increased genera,on of reac,ve oxygen species (ROS). The level of p53 ac,vity increases. mitosis declines apoptosis of cells increases replica,ve senescence increases The anatomy and func,on of organs such as the liver and heart show the degenera,ve changes of age. Cell (1997) 91:2534 Mice Terc -/- Curr Biol. (2000) 10:1459-62 Mice TerT -/-

Reac,va,on of telomerase in aged mice reverses many signs of aging

The mice were made by "knocking-in" a gene that
prevents any expression of telomere reverse transcriptase (TERT) unless an ac,va,ng drug is given to the animal. Without the drug, these mice live half as long as normal, and as they get older, they display many signs of aging:

their telomeres get shorter leading to chromosome aberra,ons; their cells undergo early replica,ve senescence; almost all their organs testes, spleen, intes,ne, brain show degenera,ve changes typical of aging. Nature (2011): 469, 102106

BUT,
if given the ac,va,ng drug over a four-week period at a

,me when degenera,ve changes were already apparent (25-30 weeks), their deteriora,on stopped and even par,ally reversed.

the length of their telomeres increased; replica,ve senescence was delayed; their life span was substan,ally increased; their brain, testes, liver, spleen, and intes,ne escaped the degenera,ve changes seen in untreated telomerase-decient mice; they produced larger lifers than untreated mice; there was reduced ac,va,on of p53, indica,ng reduced damage to their genome and reduced apoptosis in their ,ssues
Nature (2011): 469, 102106

Mitochondrial DNA replica,on

Replica,on is under relaxed control and there is no apparent accoun,ng of DNA origins that ensures that each molecule is replicated once and only once per cell cycle (a requirement assumed to be strictly enforced in the case of chromosomal DNA origins of replica,on). Consistent with this is the lack of any sharp restric,on on mtDNA replica,on with regard to cell cycle phase.

Mammalian mtDNA Replica,on Proteins

mtDNA polymerase, commonly termed DNA polymerase y There are several reports of an associated 3-5 exonuclease ac,vity being an intrinsic part of the polymerase; it likely confers a proofreading capacity consistent with studies on the error rate of DNA polymerase y The cataly,c subunit of the holoenzyme appears to be of signicant size (approximately 140 kD)

Models for mitochondrial DNA replica,on

Strand displacement Replica,on is unidirec,onal

Strand coupled Replica,on is bidirec,onal