VL 20 ( Michael Lennhard) Flashcards
key definitions
- replicon – A unit of the genome in which DNA is replicated. Each contains an origin for initiation of replication.
- origin – A sequence of DNA at which replication is initiated.
- terminus – A segment of DNA at which replication ends
An origin usually initiates bidirectional replication
- semiconservative replication
- replicated region = bubble whin nonreplicated DNA
- replication fork: initiated at origin
→moves sequentially along DNA
unidirectional:
single replication fork at origin created
bidirectional:
two replication forks at origin created
→ moves in opposite directions
10.3 The Bacterial Genome Is (Usually) a Single Circular Replicon
single origin of replication (oriC in E. coli; 245 pb)
- decatenation by DNA topoisomerases is necessary for cell division→segregate to daughter cells
- replication once per division cycle
Methylation of bacterial origin regulates initiation
oriC - contains binding sites for DnaA (initiator protein)
–> DnaA (initiator protein; unwinds ds in this region) binding sites
- 11 GATC/CTAG palindromic repeats (Dam methylase methylate A on both) strands
What happens during/after DNA replication?
* fully methylated dsDNAn → 2 hemimethylated dsDNA (original strand: methylated;
* new strand: not methylated, bc dNTPs in solution aren ́t methylated) → inactive origins
* new strand methylated by Dam methylase; 13 min delay→dsDNA fully methylated→active origin
Each Eukaryotic Chromosome Contains Many Replicons
- Eukaryotic replicons are 40 to 100 kilobases (kb) in length.
- Individual replicons are activated at
characteristic times during S phase. - Regional activation patterns suggest that replicons near one another are activated at the same time.
Replication Origins Can Be
Isolated in Yeast
- short AT sequences
- essential 11 bp sequence
- origin recognition complex (ORC): complex of 6 proteins that binds to autonomously replicating sequence;
associated with yeast origins throughout cell cycle
Licensing factor (lf) controls eukaryotic rereplication:
prior to replication:
lf in nucleus→allows DNA replication to proceed during S phase
* lf initiates replication at each origin
→lf removed, inactivated, destroyed by replication
→preventing rereplication
after replication
–> lf in nucleus inactivatedc
–> new lf may be translated in cytoplasm, but blocked from nucleus entering
during mitosis
* nuclear membrane dissolves
→lf enters nucleus + associate with nuclear material (e.g. ori)
→intiation of another replication cycle possible
DNA Polymerase
DNA Polymerases are the Enzymes that make DNA
- DNA synthesis in semiconservative replication + DNA repair reactions
- DNA Pol I: 5 ́-3 ́exonuclease activity that can be combined with DNA synthesis→nick translation
–> nick = ssDNA break in dsDNA
–> nick 3 ́-OH group = initiation site for DNA synthesis o old strand degraded by 5 ́-3 ́exonuclease - DNA Pol: 3 ́-5 ́exonuclease activity (“proofreading”)→excise incorrectly paired bases
–> E adds base to growing strand
–> wrong base inserted
→ proofreading
→ base hydrolyzed + expelled
→ reconstitute 3 ́-OH group
→new nucleotide inserted - replication fidelity improved by proofreading by factor 100
Priming is required to start DNA Synthesis
- DNA Pols require 3 ́-OH priming end
→ initiate DNA synthesis - replication priming on dsDNA requires: replicase, SSB, primase
- DnaB: helicase; unwinds DNA for replication; E. coli
Coordinating synthesis of the lagging and leading strands:
- different E units required to synthesize leading/lagging strands
- E. coli: both units contain same catalytic SU (DnaE)
- other organisms: different catalytic SU might be required for each strand
DNA Polymerase
Holoenzyme Consists of
Subcomplexes
- clamp loader: places clamp (2 β-SU), which provides processivity to E, on DNA
→ encircles dsDNA; transfer process requires ATP hydrolysis - +coreE(α,β,θ)
- τ + 2nd core E
→ symmetric dimer
- τ + 2nd core E
- at least one catalytic core associated with each template strand
→ leading/lagging strand synthesis - processivity:
E ability to perform multiple catalytic cylces with single template instead of dissociation after each cycle
The Clamp controls association of core E with DNA:
helicase DnaB:
interacts with primase DnaG→initiate each Okazaki fragment
Helicase:
* unwinds DNA; connected to DNA Pol holoenzyme
* Primase synthesizes short complementary RNA sequence
→ primase dissociates; 3 ́-OH group of RNA sequence provides priming
site
* DNA Pol III extends RNA primer→Okazaki fragment
* large ss-loop extruded→primase interacts with helicase + generates new RNA primer→new Okazaki fragment synthesis
* DNA Pol I: nick translation (nicks between Okazaki fragments)→replace RNA primer with DNA
* Ligase: seals nicks
–> E + ATP/NAD→adduct: E-AMP-complex
–> transferred to free 5 ́-P at nick site
–> cleaved diphosphate-bond
→ 5 ́-P of nick site forms phosphodiester bond between 3 ́-OH and 5 ́-P of nick o
→ ligation reactions in molecular biology
Important definitions
plasmid:
circular, extrachromosomal DNA; autonomous; self-replicating
copy number:
copy number of plasmid maintained in a bacterium (relative to copy number of origin of bacterial chromosome)
–> single-copy plasmids exist at one plasmid copy per bacterial chromosome origin
–> multi-copy plasmids exist at > 1 plasmid copy per bacterial chromosome origin
The bacterial Ti Plasmid Causes Crown Gall Disease in Plants
* In crown gall disease, infection with the bacterium Agrobacterium tumefaciens can transform plant cells into tumors.
* The infectious agent is the Ti plasmid carried by the bacterium.
* The plasmid also carries genes for synthesizing and metabolizing opines (arginine derivatives) that are used by the bacterium
classical bacterial two-component system
- Acetosyringone sensed by phosphohistidine relay (virA/virG = proteins that are encoded on Ti plasmid itself)
→ trigger synthesis of other vir proteins on Ti plasmid
→ vir proteins initate process of transferring a T-DNA copy into plant cell by ss-cut at one T-DNA borders
→ss copy of T-DNA (coated by SSB)
→nuclear protein complex (npc) into plant cells via type4
secretion system build by proteins encoded from vir genes
→nucleus
→ss
→dsDNA
→integrated into genome by host proteins
Trans-action of vir genes
→T-DNA separation and vir genes
→two plasmids:
- two functional present regions on Ti-plasmid that act in trans/cis
- vir region = trans
–> proteins act on T-DNA region → make copy of it → transfer: plant cell o don ́t care where T-DNA is located
–> example
2 T-DNA in same cell
* Mutation on one vir gene of one T-DNA→neither of the copies will be transferred into plant cell nucleus (trans)
* T-DNA = cis
–> borders can only trigger DNA transfer on which these borders are located
–> example
▪ 2 T-DNA in same cell
▪ Mutation in border sequence of one T-DNA→T-DNA with mutation will not be transferred→T-DNA without mutation
➔ helper plasmid without T-DNA but vir region + 2nd plasmid with T-DNA but without vir genes
* modifiy DNA between T-DNA borders→transfer in agrobacterium with ti-helper plasmid
* advantage: no transfer of unwanted DNA regions; easily manageable system
