DNA ReplicationDNA Replication

AHMP 5406AHMP 5406

Objectives:Objectives:

Outline the mechanisms of eukaryotic Outline the mechanisms of eukaryotic DNA replicationDNA replicationDescribe the cellular mechanisms that Describe the cellular mechanisms that help avoid error generation during DNA help avoid error generation during DNA synthesissynthesisDescribe the possible pathways of DNA Describe the possible pathways of DNA repairrepairRelate chromatin density and the cell cycle Relate chromatin density and the cell cycle to DNA replicationto DNA replication

DNA ReplicationDNA Replication

The process of copying DS The process of copying DS DNA by templated DNA by templated polymerizationpolymerization

In Eukaryotes occurs only In Eukaryotes occurs only during S phaseduring S phase

Overall replication scheme similar to prokaryotes

The process of copying DS The process of copying DS DNA by templated DNA by templated polymerizationpolymerization

In Eukaryotes occurs only In Eukaryotes occurs only during S phaseduring S phase

Overall replication scheme similar to prokaryotes

DNA ReplicationDNA Replication

Base pairing is responsible for DNA replication and repair

Multiple initiation points

Linear chromosome (Proks. circular)

Many polymerases and accessory factors required

Base pairing is responsible for DNA replication and repair

Multiple initiation points

Linear chromosome (Proks. circular)

Many polymerases and accessory factors required

Chromosome Size and TopologyChromosome Size and Topology

SpeciesGenome Size (bp)

Haploid Chromosome

Number

Chromosome Size (bp)

Chromosome Shape

Escherichia coli 5 x 106 1 5 x 106 circular

Saccharomyces cerevisiae 1.4 x 107 16 8.8 x 105 linear

Homo sapiens 3 x 109 23 1.3 x 108 linear

DNA replication is semi-conservativeDNA replication is semi-conservative

During one round of During one round of replicationreplication

One strand used as One strand used as templatetemplate

Repl. begins at specific chromosomal Repl. begins at specific chromosomal sitessites

Replication origins

Regardless of organism are: unique DNA segments with multiple short repeats

recognized by multimeric origin-binding proteins

usually contain an A-T rich stretch

Replication origins

Regardless of organism are: unique DNA segments with multiple short repeats

recognized by multimeric origin-binding proteins

usually contain an A-T rich stretch

Most DNA replication is bidirectionalMost DNA replication is bidirectional

Eukaryotic Chromosome ReplicationEukaryotic Chromosome ReplicationDNA replication are very similar in proks and DNA replication are very similar in proks and eukseuks

Differences:Differences: Euks have many chromosomes Euks have many chromosomes

one in prokaryotesone in prokaryotes The problem with nucleosomes The problem with nucleosomes

euk DNA is “packaged” euk DNA is “packaged” wrapped around histones wrapped around histones

In eukaryotes DNA and histones must be doubled with In eukaryotes DNA and histones must be doubled with each cell divisioneach cell division

Eukaryotic ReplicationEukaryotic ReplicationDNA synthesisDNA synthesis In eukaryotes In eukaryotes

small portion of the cell cycle (S)small portion of the cell cycle (S)continuously in prokaryotescontinuously in prokaryotes

Eukaryotes have more DNA to replicateEukaryotes have more DNA to replicate

How is this accomplished?How is this accomplished? Multiple origins of replication Multiple origins of replication

prokaryotes one origin – OriCprokaryotes one origin – OriC Two different polymerases Two different polymerases

Problems that must be overcome for DNA Problems that must be overcome for DNA polymerase to copy DNApolymerase to copy DNA

DNA polymerases can’t melt duplex DNA DNA polymerases can’t melt duplex DNA Must be separated for copying Must be separated for copying

DNA polymerases can only elongate a preexisting DNA or DNA polymerases can only elongate a preexisting DNA or RNA strand (the primer)RNA strand (the primer)

Strands in the DNA duplex are opposite in chemical polarityStrands in the DNA duplex are opposite in chemical polarity All DNA polymerases catalyze nucleotide addition at 3All DNA polymerases catalyze nucleotide addition at 3-hydroxyl -hydroxyl

endend Strands can grow only in the 5Strands can grow only in the 5 to 3 to 3 direction direction

Structure of DNA Rep. ForkStructure of DNA Rep. Fork Both daughter strands polymerized in Both daughter strands polymerized in 5’-3’ direction5’-3’ direction

Lagging strand DNA synth. in short Lagging strand DNA synth. in short segmentssegments Okazaki fragmentsOkazaki fragments

Proteins at the fork form a replication Proteins at the fork form a replication machinemachine

Mammalian replication forkMammalian replication fork

Specialized enzymesSpecialized enzymes

Helicases separate two parental DNA strandsHelicases separate two parental DNA strands

Polymerases synthesize primers and DNAPolymerases synthesize primers and DNA

Accessory proteins promote tight binding of enzymes to Accessory proteins promote tight binding of enzymes to DNA DNA Increase polymerase speed and efficiency (sliding Increase polymerase speed and efficiency (sliding

clamp)clamp)

Editing exonucleases work with polymerasesEditing exonucleases work with polymerases

Topoisomerases convert supercoiled DNA to the relaxed Topoisomerases convert supercoiled DNA to the relaxed formform

DNA HelicaseDNA Helicase

Hexameric ringHexameric ring

Separate DNA Separate DNA strandsstrands

Use ATP hydrolysis Use ATP hydrolysis for Energyfor Energy

PrimasePrimase

Activated by helicaseActivated by helicase

Synthesizes short RNA Synthesizes short RNA primerprimer

Uses DNA as templateUses DNA as template

Sliding clampSliding clamp

Keeps DNA Keeps DNA polymerases attached to polymerases attached to DNA strandDNA strand

Assisted by clamp Assisted by clamp loader through ATP loader through ATP hydrolysishydrolysis

Will disassociate if DNA Will disassociate if DNA pol reaches DS DNApol reaches DS DNA

Single stranded binding proteinsSingle stranded binding proteins

Bind tightly and Bind tightly and cooperatively to SS DNAcooperatively to SS DNA

Do not cover basesDo not cover bases Remain available for Remain available for

templatingtemplating

Aid in stabilizing unwound Aid in stabilizing unwound DNADNA

Prevent hairpin structuresPrevent hairpin structures

Mammalian DNA polymerasesMammalian DNA polymerases

Synthesize new Synthesize new DNA strandDNA strand

Requires primer Requires primer

DNA Pol DNA Pol Associated with Associated with

primaseprimase

DNA Pol DNA Pol Elongates Elongates

Mammalian DNA PolymerasesMammalian DNA Polymerases

: Repair and Replication and primase function

: Repair function

: Mitochondrial DNA polymerase

: Replication with PCNA (processivity factor)

: Replication

: Repair and Replication and primase function

: Repair function

: Mitochondrial DNA polymerase

: Replication with PCNA (processivity factor)

: Replication

TopoisomeraseTopoisomerase

Some proteins change topology of DNASome proteins change topology of DNA Helicase can unwind the DNA duplex Helicase can unwind the DNA duplex induce formation of supercoils induce formation of supercoils

Topoisomerases catalyze addition or removal Topoisomerases catalyze addition or removal of supercoilsof supercoils

TopoisomeraseTopoisomerase

Type I topoisomerase relax DNA by Type I topoisomerase relax DNA by nicking and closing one strand of nicking and closing one strand of duplex DNAduplex DNA

Covalently attach to DNA phosphateCovalently attach to DNA phosphate

Allow rotationAllow rotation

TopoisomeraseTopoisomerase

Type II topoisomerase change DNA topology by Type II topoisomerase change DNA topology by breaking and rejoining double stranded DNAbreaking and rejoining double stranded DNA

Action of E coli Topoisomerase I

Type II topoisomerases (gyrases) change Type II topoisomerases (gyrases) change DNA topology by breaking and rejoining DNA topology by breaking and rejoining

double-stranded DNAdouble-stranded DNA

Replicated circular DNA molecules are Replicated circular DNA molecules are separated by type II topoisomerasesseparated by type II topoisomerases

Linear daughter chromatids also areseparated by type II topoisomerases

The eukaryotic replication machinery The eukaryotic replication machinery is generally similar to that of is generally similar to that of E. coliE. coli

More on TelomeresMore on Telomeres

TelomeresTelomeres

Further evidence of a relationship b/w telomere Further evidence of a relationship b/w telomere length and aging in humanslength and aging in humans

Disorder called Disorder called progeriasprogerias (premature aging) (premature aging) Hutchinson-Gilford Syndrome (severe) – death in Hutchinson-Gilford Syndrome (severe) – death in

the teen yearsthe teen years Werner Syndrome (less severe) – death usually in Werner Syndrome (less severe) – death usually in

the 40sthe 40s

Telomere ReplicationTelomere Replication

Regions of DNA at each end of a linear Regions of DNA at each end of a linear chromosomechromosome

Required for replication and stability of that Required for replication and stability of that chromosome.chromosome.

Human somatic cells (grown in culture) divide Human somatic cells (grown in culture) divide only a limited number of times (20-70 only a limited number of times (20-70 generations)generations)

Telomere ReplicationTelomere Replication

Correlation between telomere length and the Correlation between telomere length and the number of cell divisions preceding senescence number of cell divisions preceding senescence and deathand death

Cells with longer telomeres survive longer (more Cells with longer telomeres survive longer (more divisions) than cells with short telomeresdivisions) than cells with short telomeres

Problem with TelomeresProblem with Telomeres

DNA polymerase require free 3’OH end DNA polymerase require free 3’OH end cannot replace the RNA primer cannot replace the RNA primer at the terminus of the lagging strand.at the terminus of the lagging strand.

If not remedied, the DNA would become shorter If not remedied, the DNA would become shorter and shorterand shorter

Telomerase resolves the terminal primer problemTelomerase resolves the terminal primer problem

TelomeraseTelomerase

Telomerase = enzyme made up of both protein Telomerase = enzyme made up of both protein and RNAand RNA

RNA component is base sequence RNA component is base sequence complementary to telomere repeat unitcomplementary to telomere repeat unit

Catalyzes synthesis of new DNA using RNA as Catalyzes synthesis of new DNA using RNA as templatetemplate

End-Replication ProblemEnd-Replication Problem

5

3

5

3

5

3

5

3

+

+

5

3

5

3

Process Okazaki Fragments

Telomere StructureTelomere Structure

Telomeres composed of short (6-10 bp) Telomeres composed of short (6-10 bp) repeats repeats

G-rich in one strand, C-rich in otherG-rich in one strand, C-rich in other

53

G-richC-rich

TelomeraseTelomeraseGerm-line cells possess telomerase activityGerm-line cells possess telomerase activity

Most human somatic cells lack telomerase Most human somatic cells lack telomerase activityactivity

Cultured immortal cell lines have been shown Cultured immortal cell lines have been shown to have telomerase activityto have telomerase activity

Possible cancer therapy may be to control Possible cancer therapy may be to control telomerase activity in cancer cellstelomerase activity in cancer cells