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98 Cards in this Set
- Front
- Back
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NHEJ
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nonhomologous end joining, trim then fuse DNA ends, error prone (??), KU80/KU70 end binding protein complexes w/ DNA-PK (mediates bridging), detele a couple bp's, but just do anything to get it together again.
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KU70/KU80 (euk)
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heterodimer, end-binding protein, binds dsDNA, complexes w/ DNA-PK, which mediates bridging, not known how it's removed from DNA complex.
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DNA-PK
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DNA dependent protein kinase catalytic subunit mediates briding btwn the dsDNA break
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XRCC4
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X-ray repair complementing defective repair in Chinese hamster cells 4, functions w/ DNA ligase IV and DNA-PK in repair of dsDNA break via NHEJ (bacterial = Pri)
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DNA ligase IV
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complements with XRCC 4 to fix dsDNA breaks via NHEJ (bacterial = Lig D); joins 2 ends of a dsDNA break
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Lig D
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bacterial form of DNA Lig IV in humans, which fix dsDNA breaks via NHEJ
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Homologous recombination
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ds break repair: ds break in 1 of 2 homologs is converted to a ds gap by action of endonuclease (produce 3' overhang b/c strands w/ 3' ends are degraded less than those with 5' ends). Exposed 3' end pairs with complement in intact homolog. Other strand of duplex is displaced. the invading 3' end is extended by DNA pol and branch migration, generating a DNA molec w/ 2 crossovers (Halliday intermed). Can have chromosome arms swapped or not swapped; does not lose DNA bps
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RuvC
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Resolvase that cleaves Holliday junction, binds to RuvAB complex
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RuvAB
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RuvA binds the 4 strand DNA structure formed in Holliday jxn int, and RuvB migrates strands thru each other, lets Holliday jxn move around, but doesn't require energy; loaded by RuvA
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Holliday junction
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Mobile junction btwn 4 DNA strands. Homologous sequences, so can slide up and down DNA, facilitated by RuvABC (no ATP req'd) or RecG (requires ATP)
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RecBCD
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(E.coli) aka Exonuclease V, initiates recombinational repair from dsDNA breaks, it's a helicase and a nuclease (makes ss nicks in DNA). Helicase and nucleaes activities of RecBCD degrade DNA to make 3' overhang. Chi (octameric seq, triggers helicase to start degrading only 1 strand to leave 3' overhang) pauses RecBCD, helicase alters mechanism, continues w/o nuclease. ssDNA is substrate for RecA. HUMAN = MRN
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Chi
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In homologous recomb, is an octameric seq that pauses RecBCD. Triggers helicase to start degrading only 1 strand to leave 3' overhang.
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RecA
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(E.coli) susbtrate is ssDNA (formed from RecBCD nuclease/helicase activity). Forms a filament on ssDNA; homologous duplex incorporates into complex; one of the strands in the duplex is transferred to the ssDNA orig bound to the filament; other strand of duplex displaced. (can hold ssDNA or dsDNA --> can make synapse). ATP-dependent. HUMANS = RAD51
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MRN complex
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(mammals). Mre11-Rad50-Nbs1 complex. Mammalian version of E.coli's RecBCD -- exonuclease activity to create 3' ssDNA ends. Nbs1 recognizes DSB sites. Rad51 coats 3' ssDNA region and finds homologous region on sister chromatid
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Rad51
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Mammalian version of E.coli's RecA. Coats the ss 3' ends. Dependent on BRCA1&2. ATP-dependent. Promotes strand exchange, pairing 3' end of ss with sister chromatid, DNA pol extends until 3' end can have a "sticky end" with the other 3' end of the ds break, joining it together. DNA pol and ligase fill/ligate the gap.
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nucleotide
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sugar, phosphate, base
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nucleoside
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sugar, phosphate, base
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A form DNA
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dsRNA, OH prevents B form, shorter than B form, bps wrap around helix axis (hole in middle from bird's eye view), major groove narrow and deep, minor groove wide and shallow
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B form
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right handed, bases perpendicular to backbone, 36A pitch, bps bisect helix axis
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Z form
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left handed, longer than B form
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propeller twist
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dihedral angle btwn base planes of bps; if 2 bases not coplanar, they can rock. Clashes due to propeller twist can be alleviated by bp roll/slide
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displacement
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movement of bp center away/toward helical axis. Still perpendicular but move side to side
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slide
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translation along C6 C8 axis; if bases slide out of cylinder along c6-c8 axis
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twist
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relative rotation around helix axis; how much each bp steps along helical axis
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roll
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rolling around C6-C8 axis
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tilt
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rotation of bp plane around pseudo-dyad perpendicular to twist and roll axes; like roll, but in other direction (around tilt axis)
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selve cleavage
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Rnase
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tetraloop hairpin (RNA)
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GNRA, R=purine, G and A base pair, NRA are flipped out of duplex, but stacked
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secondary structural motifs
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double strand, single nucleotide bulge, 3 nucleotide bulge, hairpin loop, symmetric internal loop, asymm internal loop, two stem jxn or coaxial stack (3 diff chains), 3 stem jxn, 4 stem jxn
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tertiary structural motifs
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pseudoknot (secondary/secondary interactions, super secondary interaction??), kissing hairpins, hairpin loop-bulge contact; non-WC bp, metal ion bridging
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Lecture 3
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Polymerases
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POLa
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DNA replication, euk
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POLb
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Base excision repair, euk
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POLg
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Mitochondrial replication, euk
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POLd
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DNA replication lagging strand, euk, displaces RNA primer, FEN1 removes RNA/DNA chimera
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POLe
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DNA replication leading strand, euk
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Klenow fragment
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DNA pol I, minus the 5' to 3' exonuclease activity. (What's left is 3' to 5' exonuclease activity, and replicative polymerase part)
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DNA Pol I
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consists of Klenow frag (3' to 5' exonuclease actvity, polymerase, and 5' to 3' exonuclease activity), cleaved by subtilisin protease
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DNA Pol III
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main replicative polym in prokaryotes, high processivity, has 3' to 5' exonuclease activity
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3' to 5' exonuclease
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back up and excise last dNTPs
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5' to 3' exonuclease
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repair (Okazaki fragment maturation)
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Polymerase structure
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fingers (dNTP sensing, did it bind correct dNTP?), palm (catalysis, has AA active site), thumb (grabs DNA)
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polymerase
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pold/e (euk), pol III (prok); requires ATP
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helicase
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DnaB (prok 5' to 3' lagging template loops thru); MCM (human 3' to 5')
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SSBs
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SSB (prok, 4mer); RPA (replicating protein A, trimer); filament is substrate for DNA pol; takes out 2ndary structure of ssDNA
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primase
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DnaG (prok, linked to DnaB helicase --> primosome, ssDNA spooled out of helicase into DnaG, dimer ensures primase is only active at replic fork rather than when there's only ssDNA); Pola/Pri1/Pri2 (pri subunits make RNA primer and handoff primer to pola, it's a chimeric RNA-DNA primer)
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clamp loader
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g/t (prok, binds 2 DNA pol IIIs to couple leading and lagging strand synth); RFC (euk, replication factor C); requires ATP; feeds RNA/DNA heteroduplex into clamp;
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clamp
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b/DnaX (prok); PCNA (euk, prolif cell nuclear agent); increases processivity b/c most DNA pols fall off strand after a short string of nucleotides, releases DNA+polym complex when it runs into a region of dsDNA
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RnaseH
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cleaves RNA primer on lagging strand and pol I fills in, OR DNA pol I uses 5' to 3' exonuclease activity to work on the previous Okazaki fragment
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FEN1
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flap endonuclease, strand displacement, migrates and chews into some DNA too
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DNA ligase
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(euk uses ATP; bacteria use NADH) ligates Okazaki frags in lagging strand, encircle DNA, looking for nicks by deformability in DNA strand, ligation straightens DNA, promotes dissociation
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linking number
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integer # of times one strands wraps around the other
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twist
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#bp divided by DNA pitch; right handed means + twist
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writhe
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if no supercoiling Wr = 0; Wr > 0 means left handed helix if Tw rt handed; Wr < 0 means right handed helix if Tw rt handed
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Type I topoisomerase
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IA (cuts ssDNA region and migrates 2nd strand before ligating, lk # changes by 1, form covalent intermed w/ 5' end of DNA); IB (nicks DNA, swivelase, DNA rotates along single phosphodiester bond, forms covalent intermed w/ 3' end of DNA)
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Type II topoisomerase
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cuts DNA duplex, linking number changes by 2; Tyr -OH attacks PO4 and forms a covalent intermed, opens gap 20Angstroms; needs ATP to load DNA segment onto topoisomerase complex
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Topoisomerase cleavage rxn
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reversible, no energy needed, uses phosphoTyr intermed. Type IB (3' cleavage, no metals); Type IA, II (5' cleavage, Mg++ assisted, but Type II needs ATP to get DNA in enzyme)
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Lecture 6
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Replication Challenges II: starting and stopping replication
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DUE
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DNA unwinding element; part of E.coli's OriC, opening point of DNA, AT rich
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ARS
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autonomous replicating sequence in yeast, contains Ori
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ChIP
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chormatin immunoprecipitation; antibody pulldown of protein you think will be bound to DNA
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DnaA
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(E.coli) recognizes Ori sequence (wraps DNA to be negatively supercoiled, may help melt DUE); opens duplex at specific sites in origin; requires ATP, dissociates when ADP dissociates; recruits helicase
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DnaC
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helicase (DnaB) loader, dissociates once DnaB is bound to DNA, ATPase
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Hda (kinase)
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(E.coli) recruited by clamp, causing ATP hydrolysis and DnaA dissociation.
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eukaryotic initiation
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ORC (origin replic complex) loads MCM helicase; Cdc6 (ATPase) and Cdt1 bind to regulate MCM assembly, pre-RC (pre-replic complex) formed, Cdt1/Cdc6 lost, GINS/Cdc45 binds MCMs, RC complex formed with primase, polym, clamps.
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Tus protein
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binds Ter sequences and inhibits DnaB helicase progression, but only in one direction, (cytosine binds Tus and locks it down so helicase can't go past it), this system is not preserved in E.coli
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Fork convergence tangle (bact)
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Resolution; replicate and use type II topo; or use type IA topo then replicate
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Replic prob in euks
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in lagging strand synthesis, after RNA primer removal, primase cannot add a new primer, so chromosome ends shorten. Releases Telomere Binding Proteins (TBPs). Further shortening affects expression of Telomere-Shortening Sensitive Genes (TSSGs).
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Lecture 7
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Fixing the mistakes, part I - repairing DNA
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depurination
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add H2O, gives depurinated sugar
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deamination
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add H2O, NH3 leaves, cytosine to uracil
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pyrimidine dimers (C and T)
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causes DNA lesion, fixed by photolyase, has 2 FADH2's, requires visible light, uses e- shuttling to break bond.
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DNA glycosylase
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base excision repair, cleave base and leave sugar intact. AP (apurinic or apyrimidinic) endonuclease fills in gap.
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DNA damage via alkylation
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Me on N of Ade or Cyt fixed by AlkB; -Me on O of Guanine fixed by O6-methylguanine methyltransferase
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BER
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(base excision repair) DNA glycosylase recognizes damaged base and cleaves btwn base and deoxyribose, AP endonuclease cleaves phosphodiester backbone near AP site, DNA pol I initiates repair from free 3'OH at nick, removing a portion of the damaged strand (uses 5' to 3' exonuclease activity) and replaces w/ undamaged DNA, nick sealed with DNA ligase
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Nucleotide Excision Repair (E.coli)
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DNA lesion (pyrimidine dimer), UvrA senses bulky lesion, UvrB and UvrC cut 13mer DNA (exinucleases), UvrD unwinds(helicase), PolI fills in, ligase seals
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UvrA
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senses bulk in dsDNA due to DNA lesion
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UvrB, UvrC
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exinucleases, cut DNA surrounding lesion (13mer in E.coli, 29mer in humans)
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UvrD
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helicase, unwinds after UvrB and UvrC cut region surrounding DNA lesion
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Nucleotide Excision Repair (humans)
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initial damage recognition by XP-C and 23B (like UvrA), TFIIH (transcription factor composed of helicases/endonucleases XPD and XPB), RPA (replic factor A helicase) stabilizes other strand, DNA pold/e fill in gap, assisted by PCNA, seal w/ DNA ligase
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E.coli methylation
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MutH binds Me-GATC, MutS binds mismatch, MutL links MutH and MutS (via DNA loop), MutH cleaves unmethylated strand on 5' side of G in GATC sequence; DNA helicase, exonuclease and SSB removes a segment of the new strand btwn cleavage site and just a lil beyond mismatch, gap filled in by DNA polIII, sealed by DNA ligase; requires 2 ATPs
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MSH
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(euk) MutS homologs, defects in mismatch excision repair lead to colon cancer; MSH2:MSH6 complex binds the mismatch and identifies newly synthesized strand. MLH1 endonuclease and other factors like PMS2 bind and recruit a helicase and exonuclease, which together remove several nucleotides, including the lesion, gap filled and sealed by Pold
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PMS2
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Mut L homolog
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MutH
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binds me-GATC, activates MutL, cleaves umethylated strand on 5' side of G in GATC
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MutS
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binds mismatch, linked to MutH by MutL (forms a DNA loop)
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TLS
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(translesion synthesis) allows DNA replic machinery to replicate past DNA lesions, switches out regular DNA pol for specialized translesion pol (DNA pol V E.coli), with larger active site that can facilitate insertion of bases opposite damaged nucleotides, low fidelity, but efficient at inserting correct bases opposite specific types of damage. put a mistake down and fix later b/c don't want ot impede replication
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histones
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conserved, 4 core histones: H2A, H2B, H3, H4, helix-turn-helix domain, 147 bp wrapped almost 2x around histone; tails emanate btwn DNA gyres--allowing for post-translational chemical modification by other enzymes
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H3/H4
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dimer forms tetramer, then assembles with H2A/H2B dimer (cannot form stable tetramer alone)
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H2A/H2B
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dimer assembles w/ H3/H4 b/c cannot form stable tetramer alone
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histone modification
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"epigenetic": the marks alter gene fxn, not part of DNA seq but control gene expression (DNA trace)
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heterochromatin
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histones are hypo-acetylated; Sir2 is a histone deacetylase (removes -OAc), Sir complex (Sir2,3,4) bind cooperatively to de-acetylated chromatin, tightly packing 30nm filament, combined actions of packaging proteins and deacetylase promote heterochromatin spreading and gene silencing; replicated later in S phase
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HAT
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histone acetyl transferase: scaffold code reader complex recognizes covalent modifications on the histone tail and attracts other proteins, leading to attachment to other components in the nucleus, leading to gene expression, silencing, or other gene fxn
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HDAC
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histone deacetylase: histone modifying enzyme modifies histone, then a code reader protein comes and causes the adjacent histone to be modified (reader-writer complex)
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histone chaperones
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control deposition of histones during replication. H3-H4 dimer stays on DNA during replication (H2A-H2B dissociate); CAF-1 loads a new H3-H4 dimer onto new strand, NAP-1 loads H2A-H2B dimer onto strands, where existing H3-H4 already exists
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CAF-1
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loads a new H3-H4 dimer onto new strand
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NAP-1
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loads H2A-H2B dimer onto strands, where existing H3-H4 already exists
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histone remodeling complex
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control nucleosome placement and exchange of octamer subunits; chaperone binds to the subunit to be replaced
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euchromatin
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transcribed and less condensed
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