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