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DNA Replication and Repair

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bidomaxo's version from 2017-10-25 05:50

Section 1

Question Answer
Initiation - Origin of Replication - ProkaryoteOrigin of Replication - 3 AT-rich sites followed by 5 specific binding sites for DnaA hexamer protein. DnaA binds 5 recognition sequences, forming hexameric coiled DNA complex.
Initiation - DNA Strand Separation - ProkaryoteHelicase Loader DnaC binds to DnaA and loads DNA helicase (DnaB) onto origin of replication. DnaB begins unwinding dsDNA that is trapped by ssDNA binding proteins (SSB) to prevent re-annealing.
Elongation - Leading Strand - Primer - ProkaryoteDnaG converts "prepriming complex" to "primosome". RNA polymerase that synthesizes short stretch of "RNA Primer" complementary and antiparallel to DNA template
Elongation - Leading Strand - Elongation - Prokaryotepol III. 5'->3' polymerase synthesizes complementary strand continuously in direction of advancing replication fork. Reads template DNA in 3'->5' direction
Elongation - Leading Strand - Primer Excision - Prokaryotepol I. 3'->5' exonuclease proofreads and excises mispaired bases. 5'-3' exonuclease excises RNA primer.
Elongation - Leading Strand - Gap Filling - Prokaryotepol I. 5'-3' polymerase fills gaps with DNA, not continuous. DNA Ligase catalyzes 3'-5' phosphodiester bonds between original DNA strand and gap filled DNA strand
Elongation - Lagging Strand - Primer - ProkaryoteDnaG converts "prepriming complex" to "primosome", RNA polymerase that synthesizes short stresches of RNA Primers (5'-3' direction) complementary and antiparallel to DNA template read in the 3'-5' direction
Elongation - Lagging Strand - Elongation - Prokaryotepol III. 5'-3' polymerase extends the chain discontinuously in the direction away from the advancing fork. Creates Okazaki fragments as new RNA primers are needed to begin the next segment of elongation. 3'-5' exonuclease proofs and excises mispaired bases
Elongation - Lagging Strand - Primer Excision - Prokaryotepol I. 5'-3' exonuclease excises RNA primers (multiple)
Elongation - Lagging Strand - Gap Filling - Prokaryotepol I. 5'-3' polymerase fills gaps left in DNA. 3'-5' exonuclease proofreads during gap filling. DNA ligase catalyzes 3'-5' phosphodiester bonds between original DNA strand (pol III) and gap filled strand (pol I)
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Section 2

Question Answer
Initiation - Origin of Replication - EukaryoteMultiple origins of replication with less well defined AT-rich regions. Origin of replication complex - hexameric protein binds to 5 sites forming coiled DNA complex required to recruit helicase and loaders
Initiation - DNA Strand Separation - EukaryoteHelicase Loaders Cdc6/Cdt1 bind to ORC, recruit DNA helicase Mcm2-7 which forms hexameric complex that unwinds dsDNA for trapping by SSB repA, which binds to ssDNA to prevent re-annealing.
Elongation - Leading Strand - Primer - Eukaryotepol alpha, primase. converts "prepriming complex" into "primosome". Synth short RNA Primer (3'-5') complementary and antiparallel to DNA template in 5'-3' direction.
Elongation - Leading Strand - Elongation - Eukaryotepol alpha DNA Polymerase synth ~20 nt in 5'-3' direction then dissociates. pol epsilon DNA Polymperase replaces alpha and synthesizes in 5'-3' direction continuously.
Elongation - Leading Strand - Primer Excision - EukaryoteFEN1 & RNase H excise RNA Primer. pol epsilon 3'-5' exonuclease proofread and excise mispaired bases.
Elongation - Leading Strand - Gap Filling - Eukaryotepol epsilon, 5'-3' polymerase fills gaps. DNA ligase catalyzes 5'-3' phosphodiester bonds to connect original strand and gap filled strand
Elongation - Lagging Strand - Primer - Eukaryotepol alpha, primase. Converts "prepriming complex" to "primosome" Synth short RNA primers (5'-3') complementary and antiparallel to the template DNA (3'-5')
Elongation - Lagging Strand - Elongation - Eukaryotepol alpha DNA polymerase synth ~20 nt in 5'-3' direction, then dissociates. pol delta then synthesizes discontinuously away from helicase. Forms RNA-DNA Okazaki fragments as DNA unwinds
Elongation - Lagging Strand - Primer Excision - EukaryoteFEN1 & RNase H excise RNA primers. pol delta 3'-5' exonuclease proofreads and excises mismatched bases
Elongation - Lagging Strand - Gap Filling - Eukaryotepol delta, 5'-3' polymerase fills gaps. DNA ligase catalyzes 5'-3' phosphodiester bonds between original strand and gap filled segments.
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Section 3

Question Answer
Prokaryotic Replication - Cell CycleProkaryotic DNA = circular plasmic attached to plasma membrane. Replicated dsDNA contains one original strand (SEMICONSERVATIVE) with unique point of attachment to cell membrane - allows cell fission to occur.
Eukaryotic Replication - Cell CycleDNA replication occurs during the S "Synthesis" Phase of the cell cycle
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Prokaryotic Replication - SupercoilingDNA Gyrase relaxes coils in prokaryotic DNA during replication. It is a topoisomerase II enzyme that can break and reseal a DNA strand.
Eukaryotic Replication - Telomeres - Chromosomal EndsChromosome terminates with ds hexameric repeat 5'-AGGTT-3' then a G-rich 3' overhand. The overhang folds over on self with the help of telomere binding proteins (TBP) to prevent chromosomal degradation by exonucleases
Eukaryotic Replication - Telomeres - End ShorteningWhen RNA primer excised from 5' end of lagging strand, the gap cannot be filled with DNA, causes end to be shortened. This shortening continues with further replication, creating "mitotic clock" counting down to a critical length inhibiting replication called senescence
Eukaryotic Replication - Telomeres - End Lengtheningtelomerase "reverse transcriptase" 5'-3' RNA-dependent DNA polymerase used to lengthen telomeres by repeated addition of ssDNA hexameric AGGGTT to 3' end of DNA. The enzyme contains the complementary C-rich RNA sequence that will selectively bond DNA nucleotides to the 5' end of the strand, then translocate further down. Regular DNA elongation (leading and lagging) then occur. Called "immortilization".
DNA Damage - Base AlterationsDeamination - exocyclic amino groups in pyrimidine rings (A, G, C) undergo spontaneous hydrolysis, generating a carbonyl and releasing ammonia
DNA Damage - Intrastrand Cross LinkingThymine Dimers - UV causes covalent cross linking between adjacent thymines on same DNA strand
DNA Damage - Interstrand Cross LinkingPsoralen - poison that intercalates into dsDNA and, w/ UV, crosslinks thymines on opposite strands
DNA Repair - Base Alterations and Mismatches - Base Excision Repair (BER)Activated by Deamination of A, G, C and losses of single bases. Recognition, Base excision (glycolases), Ribose excision (ribose (strand) cleaved), DNA synthesis, Strand ligation.
DNA Repair - Base Alterations and Mismatches - Nucleotide ExcisionActivated by intrastrand thymine dimers and mismatches from errors in proofreading. Recognition, Strand nick (endonucleases cut on both sides of nt), strand excision (exonuclease removes bad strand), DNA synthesis (DNA polymerase), Strand ligation
DNA Repair - Double Strand Breaks - Non-Homologous End Joiningbroken ends of dsDNA directly ligated as long as there are adequate ss overhands to re-anneal. Becomes error prone as overhangs damaged/removed
DNA Repair - Double Strand Breaks - Homologous Recombinationgenetic recombination in which nucleotide sequences are exchanged between two similar or identical strands of DNA. Resection (5' ends degraded to create 3' overhang), Strand invasion (3' overhang of broken DNA invades and pairs with complementary strand of non-broken DNA), Second-end capture (displaced unbroken strand pairs with 3' overhang of other broken strand), DNA synthesis (using invaded strands as templates, 3' overhang extended), Branch migration (to continue synthesis, intersection of strands continues to move in same direction), Resolution (regenerated full sequence strands dissociate and re-anneal)
PCRDNA Amplification. Template DNA strands heat separated, Primer DNA (short 5' sequences complementary with 3' end of each template strand flanking desired target region) annealed at low T, 5'-3' DNA synthesis by Taq polymerase at high T, & repeat
DNA Repair - Strand RecognitionProkaryote - Parental strand is methylated. Mismatched bases cleaved from unmethylated strand. Eukaryotes - method not understood.
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