Regulation of DNA replication

Question 1

E.coli orgin of replication

Answer 1

Have three 13-mer direct repeats that are AT rich and are the locus of initial DNA strand separation
Have four 9-mer DNA sites that bind DnaA initiator protein
also contains 11sites that are methylated

Question 2

describe the replication initiation at OriC in bacteria

Answer 2

The initiator protein (DnaA) binding oriC with limited parental strand separation

Initial strand separation at AT-rich site in oriC establishes a loading site for the replicative helicase (DnaB) which is loaded by the helicase loader (DnaC).

Expansion of the melted region by DnaB, stabilization of the melted region by SSB followed by primer synthesis by primase (DnaG) and replication fork establishment by clamp loading and recruitment of DNA pol III holoenzyme

Question 3

How is the initiation at the E. coli origin regulated? (2 ways)

Answer 3

1. Newly replicated oriC DNA is hemimethylated (only on the parental strand) and this form the the origin is sequestered by SeqA preventing DnaA from rebinding
2. DnaA hydrolyses the bound ATP to ADP and the oriC converts to the closed conformation. Exchange of ADP for ATP by DnaA is slow (30 min) allowing time for cell division to finish

Question 4

T/F in ideal growth conditions bacterial origins can ‘fire’ multiple times during the cell division cycle resulting in higher copy number of genes near the origin than genes distant from the origin of replication.

Answer 4

True

Question 5

Why do eukaryotes need multiple origins

Answer 5

Replication in eukaryotes is much slower so multiple origins (spaced 10 to 40 kb apart) are needed to replicate the large chromosomes of eukaryotes

Question 6

How often do origins fire in eukaryotes

Answer 6

once per cycle

Question 7

order of cell cycle

Answer 7

G1-S-G2-M

Question 8

Describe the ploidy in phases of the cell cycle and time of each (24 hours total)

Answer 8

G1 phase: diploid cells(6-12 hours)
S: tetraploid(6-8 hours)
G2: Tetraploid(3-4 hours)
M: Duplicated chromosomes are divided equally and the cell splits into two daughter cells, each 2n(1 hour)
G0: quiet state where it doesn’t go under division

Question 9

describe the initiation of replication in eukaryotes

Answer 9

The prereplication complex (preRC) assembles in G1 phase (middle).

The initiator, ORC, binds to the conserved A element and the B1 element.

MCM helicases are loaded onto the DNA by Cdc6 and Cdt1.

Question 10

How is progression through the cell cycle regulated

Answer 10

regulated by cyclin-dependent protein kinases

Question 11

What is a replicon

Answer 11

the length of DNA replicated from a single origin (for bacteria this is the whole chromosome or plasmid)

Question 12

What is an initiator protein

Answer 12

a protein that binds specific sites in an origin of replication and serves as a nucleation site for the assembly of other protein complexes necessary to initiate replication; for example, DnaA in E. coli, and ORC in eukaryotes.

Question 13

What are the initiator proteins in E. coli and eukaryotes

Answer 13

DnaA in E. coli, and ORC in eukaryotes.

Question 14

What is an open complex

Answer 14

a complex assembled on the E. coli origin of replication, oriC, at an early stage of replication initiation. It includes an oligomer DnaA, ATP

Question 15

What is the Dam methylase

Answer 15

an enzyme of E. coli that methylates adenine residues in the palindromic sequence GATC on both strands of the DNA.

Question 16

What is the prepriming complex

Answer 16

complex of proteins assembled at oriC in E. coli at an early stage of replication fork assembly (includes a DnaA oligomer bound to the DNA and DnaB helicases stabilizing the single strands of DNA in the replication “bubble”).

Question 17

What is the Origin recognition complex

Answer 17

origin recognition complex (ORC): a eukaryotic initiator protein complex that assembles at an origin to initiate replication

Question 18

What is the prereplication complex

Answer 18

the complex of proteins, including the origin recognition complex (ORC) and MCMs, that assembles during the G1 phase of the eukaryotic cell cycle, thereby marking the origin for replication during S phase.