Islamic University Faculty of Medicine 2012 2013

Islamic UniversityFaculty of Medicine

2012‐2013

• Melting of the two strands polymerization of new complementary strands.

• Decisions of when, where, and how to initiate replication

• Only one complete and accurate copy of the genome is made before a cell divides.

Molecular Biology, 2012‐20132

• Semiconservative mechanism of DNA replication visually verified in 1963 using autoradiography.

• Bidirectional replication of the E. coli chromosome.

• One origin of replication.• Replication intermediates are termed theta () structures.


• Can only add nucleotides in the 5′→3′ direction.• Cannot initiate DNA synthesis de novo.• Require a primer with a free 3′‐OH group at the end.

• dNTPs are added one at a time to the 3′ hydroxyl end of the DNA chain.


• A dNTP added is determined by complementary base pairing.

• Two terminal phosphates are lost the reaction is irreversible.


• Problem –DNA polymerases can only add nucleotides from 5′→3′ but, the two strands of the double helix are antiparallel.

• Solution– Semidiscontinuous replication.


• Major form of replication in eukaryotic nuclear DNA, some viruses, and bacteria.


• Once primed, continuous replication is possible on the 3′→ 5′ template strand (leading strand).

• Synthesis occurs in the same direction as movement of the replication fork.


• Discontinuous replication occurs on the 5′→3′ template strand (lagging strand).

• DNA is copied in short segments called “Okazaki fragments” moving in the opposite direction to the replication fork.


• Synthesis of both strands occurs concurrently– Nucleotides are added to the leading and lagging strands at the same time and rate.

– Two DNA polymerases, one for each strand.

• Fundamental features of DNA replication are conserved from E. coli to humans.


• DNA polymerase I– Primer removal, gap filling between Okazaki fragments, and nucleotide excision repair pathway.

– Two subunits: Klenow fragment has 5′→3′ polymerase activity; other subunit has both 3′→5′ and 5′→3′ exonuclease activity.

– Unique ability to start replication at a nick in the DNA sugar‐phosphate backbone.

– Used extensively in molecular biology research.


• DNA polymerase III– Main replicative polymerase.

• DNA polymerase II– Involved in DNA repair mechanisms.

• DNA polymerases IV and V– Mediate translesion synthesis


• An origin of replication is a site on chromosomal DNA where a bidirectional replication fork initiates or “fires.”– Usually A‐T rich.– In E. coli the initiator protein DnaA can only bind to negatively supercoiled origin DNA.

• Most bacteria have a single, well‐defined origin (e.g. oriC in E. coli)

• Some Archaea have as many as three origins (e.g. Sulfolobus).


Helicase: unwinds the parental double helix.

Sliding clamps: tether DNA polymerase to the DNA

Clamp loader that uses ATP to open and close the sliding clamps around the DNA

Primase that initiates lagging strand Okazaki fragments

Single‐strand DNA binding proteins (SSB): protect the DNA from nuclease attack.

• New clamps are assembled; DNA polymerase III hops aboard to make the next Okazaki fragment.

• This process occurs around the circular genome until the replication forks meet.

• In E. coli, the replication forks meet at a terminus region containing sequence‐specific replication arrest sites.


• DNA polymerase I removes the RNA primers and replaces them with complementary dNTPs.

• DNA ligase catalyzes the formation of a phosphodiester bond between adjacent Okazaki fragments.

• Movement of the replication fork machinery results in:– Positive supercoiling ahead of the fork.– Negative supercoiling in the wake of the fork.– Torsional strain that could inhibit fork movement is relieved by DNA topoisomerase.


• DNA polymerase III can be blocked by a damaged site on the template DNA.

• Sometimes DNA polymerase collides with RNA polymerase and is stalled.

• In both cases, replication can be jumpstarted on the leading strand by formation of a new primer at the replication fork.


• Chromosomal DNA replication– DNA pol , pol , pol

• Mitochondrial DNA replication– DNA pol

• Repair processes – All the rest


• Internal sites on linear chromosomes.– Mice have 25,000 origins, spanning ~150 kb each.– Humans have 10,000 to 100,000 origins.– In the budding yeast Saccharomyces cerevisiae there is a consensus sequence called an autonomous replicating sequence (ARS).

• Mammalian origin sequences are usually AT rich but lack a consensus sequence.


• Number of origins used & the rate at which they initiate overall rate of replication – During early embryogenesis, origins are uniformly activated.

– At the mid‐blastula transition, replication becomes restricted to specific origin sites.


• Replication forks are clustered in “replication factories.”

• Forty to many hundreds of forks are active in each factory.


• Loosening of the chromatin to allow disassembly of the nucleosomes and access to the template DNA– Histone modifications (particularly acetylation)– chromatin remodeling factors


• DNA replication is restricted to S phase of the cell cycle.

• Origin selection is a separate step from initiation.– Formation of a prereplication complex.

• Prevents overreplication of the genome.


• The ATP‐dependent origin recognition complex (ORC) binds origin sequences.– The SV40 T antigen functions as a viral ORC.


• ORC recruits Cdc6 and Mcm proteins.• ORC also loads the licensing protein complex, Mcm2‐7.– Mcm2‐7 is a hexameric complex with helicase activity.


• Only licensed origins containing Mcm2‐7 can initiate a pair of replication forks.

• ATP hydrolysis by ORC stimulates prereplicationcomplex assembly.


• DNA helicases are enzymes that use the energy of ATP to melt the DNA duplex.

• Mcm2-7 helicase is bound to the leading strand template and moves 3′→5′.

• In eukaryotes, the RNA primer is synthesized by DNA polymerase (pol) and its associated primase activity.

• The pol /primase enzyme synthesizes a short strand of 10 bases of RNA, followed by 20‐30 bases of initiator DNA (iDNA).


• The hand‐off of the DNA template from one polymerase to another.


• Leading strand:– switch from DNA polymerase to pol

• Lagging strand:– switch from pol to pol

• Polymerase switching is regulated by PCNA.• Once DNA pol is recruited to the leading strand, synthesis is continuous.

• Lagging strand synthesis requires repeated cycles of polymerase switching from DNA pol to pol .


• PCNA: proliferating cell nuclear antigen.• Plays an important role in many cellular processes.

• In DNA replication, acts as a sliding clamp to increase DNA polymerase processivity.


• Replicative polymerases are high fidelity but not perfect:– 10‐4 to 10‐5 errors/bp.

• Proofreading exonucleaseactivity – 10‐7 to 10‐8 errors/bp.

• DNA polymerase has a hand‐shaped structure.– 5′→3′ polymerase activity is within the fingers and thumb.

– 3′→5′ exonuclease activity is at the base of the palm.


• The abnormal genometry of mismatched base pairs results in steric hindrance at the active site.

• Base‐base hydrogen bonding also contributes to fidelity.


• RNA primer removal.• Gap fill‐in.• Joining of Okazaki fragments on the lagging strand.


• Ribonuclease H1 nicks the RNA primer and the primer is degraded by FEN‐1 (flap endonuclease 1)

• DNA pol causes strand displacement and FEN‐1 removes the entire RNA containing 5′ “flap.”


• The remaining gaps left by primer removal are filled in by DNA polymerase or .


• Nicks are sealed by DNA ligase I.

• In association with PCNA, DNA ligase I joins the Okazaki fragments by catalyzing the formation of new phosphodiester bonds.

• Nucleosomes re‐form within approximately 250 bp behind the replication fork.

• Chromatin assembly factor 1 (CAF‐1) brings histones to the DNA replication fork in association with PCNA.

• Histones H3 and H4 form a complex and are deposited first, followed by two histone H2A‐H2B dimers.


• In eukaryotes, replication continues until one fork meets a fork from the adjacent replicon.

• The progeny DNA molecules remain intertwined.• Toposiomerase II is required to resolve the two separate progeny genomes.


• Target rapidly growing cells.• Act either as inhibitors of at least one step in the catalytic cycle or as poisons.

• Topoisomerase I is a target for a number of anti‐cancer drugs.– e.g. camptothecin


• When the final primer is removed from the lagging strand, an 8‐12 nucleotide region is left unreplicated.

• Predicts that chromosomes would get shorter with each round of replication.


• Eukaryotic chromosomes end with tandem repeats of a simple G‐rich sequence.– Humans: TTAGGG– Tetrahymena: TTGGGG

• Seal the ends of chromosomes.

• Confer stability by keeping the chromosomes from ligating together.


• The enzyme telomerase: A ribonucleoprotein(RNP) complex with reverse transcriptaseactivity.– RNA: Telomerase RNA component (TERC)

– Protein: Telomerase reverse transcriptase (TERT)


• Telomerase elongates the 3′ end of the template for the lagging strand (G‐rich overhang).

• Repeated translocation and elongation steps results in chromosome ends with an array of tandem repeats.


• Elongation of the shorter lagging strand (C‐rich strand) occurs by the normal replication machinery.

• Alternatively, the 3′ overhang folds into a t‐loop structure, which prevents telomerase access.

• Telomerase‐mediated telomere maintenance is widespread among eukaryotes from ciliates to yeast to humans.

• A striking exception is the fruitfly Drosophila melanogaster, which maintains telomeres by the addition of large retrotransposons.

• In human and fungi, telomeres can also be maintained by a recombination‐based mechanism.


• Telomere length regulation involves the accessibility of telomeres to telomerase.

• Length control involves a telomere‐specific protein complex formation – When the telomere is long enough The action of telomerase is blocked.

– When the telomere is too short Telomerase is no longer inhibited.


• A model for t‐loop formation– The 3′ single‐stranded DNA tail invades the double‐stranded telomeric DNA.

– A loop forms in which the 3′ overhang is base paired to the C strand sequence.

– The t‐loop may aid in preventing telomerase access.


• In most unicellular organisms, telomerase has a “housekeeping function.”

• In most human somatic cells, not enough telomerase is expressed to maintain a constant telomere length Progressive shortening of telomeres.

• High levels of telomerase activity in ovaries, testes, rapidly dividing somatic cells, and cancer cells.


• The Hayflick limit is the point at which cultured cells stop dividing and enter an irreversible state of cellular aging (senescence).– Proposed to be a consequence of telomere shortening.


• Telomerase: A target for anti‐aging therapy or anti‐cancer therapy?

• Cellular senescence may be a mechanism to protect multicellular organisms from cancer.

• Cancer cells become immortalized and thus can grow uncontrolled.

• In most cancer cells, telomerase has been reactivated.


• Premature aging syndrome.• Problems in tissues where cells multiply rapidly and where telomerase is normally expressed.

• Two forms of dyskeratosis congenita:– X‐linked recessive– Autosomal dominant


• X‐linked recessive dyskeratosis congenita– Mutations in dyskerin gene.– Dyskerin is a pseudouridine synthase that binds to small nucleolar RNAs and to telomerase RNA.

– Patients with dyskerin mutations have 5‐fold less telomerase activity than unaffected siblings.

• Autosomal dominant dyskeratosis congenita– Mutations in telomerase RNA gene – Partial loss of function of telomerase RNA.


• Inhibition of liver cirrhosis in mice by telomerase gene delivery.

• Why hasn’t this gene therapy strategy progressed to human trials?


Islamic University Faculty of Medicine 2012 2013

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