DNA replication

Question 1

Semi-Conservative

Answer 1

following replication, each newly replicated DNA double helix contains one intact parental strand and one newly synthesized daughter strand.

Question 2

Bidirectional

Answer 2

replication begins at a site named replication origin and simultaneously moves out in both directions from this origin. Eukaryotes have multiple origins of replication

Question 3

Okazaki fragments

Answer 3

The lagging strand of DNA is formed by discontinuous copying of the parental strand that runs 3´- to-5´ away from the replication fork. As more of the helix is unwound, synthesis of the lagging strand begins from another primer. The short fragments (100-200 bp in eukaryotes and 1000-2000 bp in prokaryotes) formed by this process are known as Okazaki fragments.

Question 4

Origin

Answer 4

Region where DNA replication begins. Contain unique short repeats. AT rich. Eukaryotes have multiple, bacteria one

Question 5

Fork

Answer 5

Forks are the sites at which DNA synthesis is occurring.

Question 6

Origin recognition complex (ORC)

Answer 6

consists of 6 protein subunits and binds to the origin of replication. Once recruits Cdc6 and Cdt1, becomes pre-RC. Before replication can initiate, it must be activated by kinases and then it recruits DNA helicase.

Question 7

DNA helicases

Answer 7

Unwinds the parental strands.

Question 8

Single-strand binding proteins

Answer 8

bind to single strand of DNA and hold in single-stranded conformation. Also protects strand from nuclease attack.

Question 9

Primase

Answer 9

lays down 10-20 bp RNA primer which DNA polymerase can bind to

Question 10

DNA polymerase alpha

Answer 10

Contains DNA polymerase and primase activity. Does not have proof-reading activity. DNA Pol α synthesizes the first ~20 deoxyribonucleotides after the RNA primer, and is then swapped for Pol δ and Pol ε for lagging and leading strand synthesis, respectively.

Question 11

DNA polymerase delta

Answer 11

DNA polymerase that synthesizes okazaki fragments

Question 12

DNA polymerase epsilon

Answer 12

DNA polymerase that synthesizes leading strand

Question 13

DNA ligase

Answer 13

catalyzes formation of phosphodiester bonds between 3´-hydorxyl group and 5´-phosphate group. Fuses okazaki fragments.

Question 14

Sliding clamp (PCNA)

Answer 14

Associated w/ eukarytoic DNA polymerase delta. Keeps DNA polymerase tightly associated with parent increasing its processivity.

Question 15

Topoisomerase

Answer 15

prevents supercoiling of the DNA at the fork. Perform by breaking and rejoining DNA strands.

Question 16

Telomerase

Answer 16

a reverse transcriptase that uses an RNA template to copy DNA repeats onto the ends of telomeres.

Question 17

Reverse transcriptase

Answer 17

RNA dependant DNA polymerase. Creates cDNA from an RNA template. Mainly associated with retroviruses which then integrate their genome into a host w/ integrase.

Question 18

Describe the order of events that occur during, the differences between, and coordination of, DNA synthesis on the leading strand and the lagging strand. (7)

Answer 18

ORC (origin recognition complex) binds to the origin of replication. Recruits Cdc6 and Cdt1, become pre-RC (replication complex). Kinase phosphorylates the complex and it recruits DNA helicase.

DNA helicase melts the parent strands bidirectionally

Single strand binding proteins (RPA euk) bind to the parent strands to protect from nuclease attack and hold it in a single-stranded conformation.

DNA polymerase alpha synthesizes the first 20ish nucleotides. It is than swapped by polymerase delta (synthesizes lagging strand) and polymerase epsilon (synthesizes leading strand)

Replisome coordinates synthesis of the leading and lagging strand by rotating the lagging strand 180 degrees.

DNA polymerase (not sure which one) removes RNA primers w/ exonuclease activity and adds dNTP’s in a 5’-3’ direction.

DNA ligase seals nicks left from the fragments.

Question 19

Describe the “end replication problem” and the activity of telomerase. (2)

Answer 19

The lagging strand cannot be synthesized to the very end because there is nothing for the last primer to bind to (in creation of okazaki fragments). Because of this, the telomeres shorten with replication until the lack of genome initiates cell death.

Telomerase is an RNA dependent DNA polymerase that maintains telomeres by copying the telomeric repeat sequence from an RNA template.

Question 20

Progeric Syndromes

Answer 20

Caused by RecQ helicase mutations
Werner’s syndrome: WRN helicase mutations
Bloom syndrome: BLM helicase mutations

Question 21

Meier-Gorlin syndrome

Answer 21

Caused by Pre-RC mutations

partial loss of function in any of the 6 subunit proteins of ORC

Question 22

FILS syndrome

Answer 22

DNA pol epsilon mutation.

Facial dysmorphism, immunodeficiency, livedo, short stature