Exam II Flashcards
Why is homologous replication so important for all organisms?
Genetic diversity and to aid the collapsing/stalling of replication forks. No need to degrade dna and start replication over.
Describe the 3 steps of recombination
2 dna molecules that are the same or similar must be broken in the same region and joined (crossover) to form two recombinant molecules.
List and explain the 4 requirements in order for homologous recombination to occur.
- identical or similar dna sequence (inter/tramolecular)
- recombination must take place in the same locus. complementary base pairing. Synapse = point of ds dna held together by base pairing
- DNA must be broken and ligated (need recombination enzymes that are nucleases and ligases)
- Heteroduplexes, which are regions where 2 complementary bp between 2 dna strands in a synapse from TWO different sources
Describe recBCD structure and function
3 subunit enzyme. Rec B and C are required for recombination. Degrades foreign dna in the cell. Helps restart replication when it stalls/fork collapses. Helps LOAD??s 😭 recA onto ssDNA. ssexo/endonuclease activity, bipolar helicase activity, ATPase activity (has to unzip to get in👀). Rec is a toxic bipolar bf
Describe the steps/process of what RecBCD does on a dna molecule
Rec LOAD??s himself onto the broken ends of a dna molecule and starts snipping off a single strand from 3’ to 5’ end. When he sees a Chi site , that activity is inhibited and he starts degrading 5’ to 3’ end. There is a 3’ overhand that gets loaded with recA to create the nucleoprotein filament. The chi site stimulates recombination. D loop formed.
What does recD do?
degrade dna
What are chi sites and why are they important
gc rich sequence found in one strand of dna. Important for recombination and protection, bc phages are unlikely to have chi sites. Without chi sites recBCD would degrade the entire dna.
What are holliday junctions?
Aint nobody going on a holiday. But anyway these form when recA-loaded ssdna invades a homologous dna molecule. These can move as no h bonds are being made or broken but it’s just complimentary base pairing.
How are Holliday junctions resolved?
ruv proteins. Ruv A, B, and C
Name the 4 types of plasmids
F (fertility), R (resistance to drugs), Col, Ti (tumor inducing in plants).
What does it mean when a plasmid is covalently closed circular?
circular plasmid folds and supercoils around itself, covalently bonded to other nucleotides. flies through agarose due to compact nature.
What are the requirements for plasmid replication?
host replication proteins (pol I, ligase). Origin of replication. Replication initiation proteins are required for the plasmid to have but other proteins are borrowed from the host.
What is the difference between oriV and oriT?
oriV is the site of replication for plasmids and oriT is the site that DNA transfer is initiated
What are the two methods of replication for plasmids?
Theta and rolling circle replication
Describe Theta replication for plasmids.
Most common, can be unidirectional (one rep fork) or bidirectional (two rep forks). An RNA primer initiates replication. Partially replicated portion and twisted unreplicated portion. When complete, one nicked DNA and one supercoiled DNA. Used by E.coli.
Describe simply the 2 steps of Rolling Circle Replication
- dsDNA->ssDNA circle and dsDNA circle
- ssDNA -> dsDNA
What is a replicon?
plasmid that can replicate independently
Describe in an extreme, nearly inhumane amount of detail, the first step of Rolling circle replication.
First, the Rep protein recognizes and binds to a palindromic sequence that contains the double-strand origin (DSO) on the DNA. Binding of the Rep protein to this sequence might allow the formation of a cruciform structure by base pairing between the inverted-repeat sequences in the cruciform. Once the cruciform forms, the Rep protein can make a nick in the sequence. Rep stays bound at 5’ end through a tyrosine. DNA pol III used 3’ OH to synthesize/build new strand while displacing one of the strands. Rep makes another cut to release displaced DNA and covalently closes displaced strand. Newly synthesized strand is closed by DNA. ligase (dsDNA).
Describe the 2nd step of rolling circle replication.
ssDNA gets synthesized into dsDNA. Uses host proteins only. Primer is made at the SSO by RNA polymerase. DNA Pol I removes primer with flap endonuclease and ligase closes the ends.
What is a host range for plasmids?
All the types of bacteria a plasmid can survive in and is determined by the ori region.
Define copy number
Average number of molecules that particular plasmid in a cell, as this must be regulated to some degree. Plasmids with high copy numbers are relaxed and need inhibited when the number reaches a certain number. Plasmids with low copy numbers are stringent .
How are relaxed plasmids regulated?
Antisense RNA
what is antisense RNA?
a piece of RNA that is complementary to a plasmid and prevents anything from binding to it to be replicated
What are par systems?
partitioning systems meant to divide plasmid copies to daughter cells after cell division
describe the F plasmid
self transmissible Fertility plasmid, best understoof
what are the 3 F plasmid cis sites/ replication origins
RepFIA (oriV), RepFIB (oriS), and RepFIC (inactivated RepA ori)
What two components are tra genes responsible for
Dtr and Mpf
What does the mpf component do
pilus, holding donor and recipient cells together, communicates with Dtr through a coupling protein
Which protein is the coupling protein for conjugation
TraD
what kind of protein is pilin for the pills made from
traA
the pills extend through what 2 proteins that span in IM/OM
tra k and tra b
what happens to plasmids that are mutant for pilin synthesis?
cannot transfer
What is the difference of outcome when a phage binds to the tip of the pilus vs the base of the pilus
tip: no transfer
base: transfer still occurs
Can you describe how antisense RNA is used for Coll P9 plasmid
After the inc region there is repY and repZ regions. With no antisense mRNA a pseudo knot is formed and translation of y and z occurs. Replication happens. With antisense RNA pairs with the loop of the upstream hairpin
