BIOL 300 Genetics Quiz 4

what are two functions of DNA polymerase III?

1. synthesize DNA (catalyze)
   
   

2. acting as a nuclease and proofreading/editing dna from the 3' - 5' direction and catalyzing dna from the 5' to 3' direction

DNA replication happens during which phase of the cell cycle?

S phase of Interphase

what are two common mistakes that dna polymerase can make?

inserting or deleting the wrong nucleotide into the strand or mismatching base pairs (substitution) and causing a frameshift error

what is a replicase? which of dna polymerases are replicases?

replicase is the enzyme that can synthesize new nucleic acid sequences from an existing template (i.e. dna polymerase III)

when is dna shifted from the polymerization domain to the exonuclease domain?

when replicated DNA contains an error/mutation;

when dna pol detects an error

frameshift mutations are rare. why?

bc they mostly occur on repetitive dna and there’s proofreading and DNA editing

what is processivity?

the ability of dna polymerase to carryout synthesis without frequent dissociations

aka

how many nucleotides can get incorporated into a daughter strand without the polymerase falling off

rna polymerase makes more mistakes than dna polymerase. why?

bc rna polymerase has no proofreading exonuclease and cant correct its own mistakes but dna pol CAN

in which direction does dna polymerase III synthesize new strands?

5' - 3'

which way does dna polymerase III read the old strands?

3' - 5'

what is the direction of the exonuclease activity of dna polymerase III?

3' - 5'

which dna polymerase has 5' to 3' exonuclease activity?

dna polymerase I

what specific action of helicase leads to opening of the dna helix?

unwinds by melting hydrogen bonds

why is primase required for dna replication?

bc primase synthesizes rna primers which provide 4' OH to polymerase

what enzyme adds the primer?

DNA primase

what enzyme removes the primer?

dna polymerase I

what enzyme seals the end where the primer was?

dna ligase

what are four enzymes that required atp at the replication fork?

dna helicase, dna gyrase, dna ligase (but primase may as well? not sure), and dna polymerase III

what is the difference between leading and lagging strands?

leading strands requires 1 primer and can be continuously synthesized while lagging strands requires multiple primers and is discontinuously synthesized with okazaki fragments

what direction is the leading strand synthesized?

5' - 3'

what direction is the lagging strand synthesized?

5' - 3'

what is the name of the segment between two primers on the lagging strand where dna synthesis occurs?

okazaki fragments

are there two polymerases, one "going backward" on the leading strand and one "going forward" on the lagging strand?

no, there is only one polymerase doing replication (dna pol III), but it has two catalytic cores for each strand

what is the difference between an F+ bacterial cell and an Hfr bacterial cell?

f+ contains a complete f plasmid wherase hfr is an f plasmid integrated into bacterial genome

can F+ cells conjugate with Hfr cells? can Hfr cells conjugate with F+ cells? can f+ cells conjugate with f- cells? can F cells conjugate with Hfr cells?

no; no; yes; yes

in what form is dna transported to the recipient cell, double- or single-stranded?

single stranded

where does the dna become double-stranded?

in conjugation: once it reaches the recipient cell

in replication: at the replication fork since DNA can be synthesized from the primers

what is the difference between a plasmid and episome?

all episomes are plasmids however not all plasmids ae episomes. this is because an episome is a plasmid that has the ability to integrate into the host chromosome

describe at least 2 tra genes and their function.

traM protein recognizes a mating pair traY protein binds near oriT and recruits traI

traA makes pili

traI makes nicks on oriT, covalently bonding to the 5’ end

when is a cell transformed from F+ or HFr?

when the F+ plasmid gets integrated into the host genome

how did Hfr get its name?

stands for "high frequency recombination"

explains how the plasmid integrates into the chromosome

what is the difference between a pilus and transfer tube?

• a pilus establishes attachment. it’s a n extension that starts conjugation by touching and is hair-like

• the transfer tube carries dna and is the actual connection bridge where transfer occurs

why are F+ cells more likely than Hfr to transfer genes required to convert F- to F+/Hfr cell?

during conjugation the connection is more likely to break since more dna (the whole chromosome) is being transferred

what part of the bacterial genome carries information that can transform a normal plant cell into a tumor cell?

the t-dna is carried on the Ti plasmid

what is one advantage that some transgenic plants have?

they are naturally resistant to some insects or pests that are harmful to them

what is agrobacterium unusual?

has Ti plasmid which causes tumors ; it can infect organisms of a different kingdom (plants) with its own DNA without entering the plant cell. it injects its own T-DNA into the plant cell

which vir genes are constitutively expressed (always transcribed)?

virA and virG

the 3D structure of DNA polymerase III (the dna bind domain) is analogous to what?

the right hand

the palm

provides the catalytic active site

fingers

position the template in active site

thumb

binds dna as it exits (processivity)

dna polymerase III exonuclease activity

resides in independent domain with its own catalytic site (3’-5’ exonuclease)

to proofread so remove nucleotides

dna polymerase III n terminal domain

spacer between catalytic and exonuclease domains

what are the four dna polymerase holoenzyme subcomplexes found within the dipolymerase replication model?

catalytic, dimerizing subunit, clamp, and clamp holder

• dipolymerase: 2 catalytic subunits

catalytic α

subunit has dna polymerase activity

catalytic ε

subunit has 3' - 5' proofreading exonuclease activity

catalytic θ

subunit stimulates the exonuclease activity

dimerizing subunit τ

links two cores

clamp β

holds the catalytic core on the dna template

• one holds leading & one holds lagging

clamp loader γ

• complex consisitng of 5 proteins

• opens the beta clamp to attach it to DNA strand

dna helicase

• unwinds dna

• 1 ATP / bp of melting

• melts H-bonds

• releases strain on molecule

dna gyrase

releases strain on molecule

• type of topoisomerase

single stranded binding proteins

• (SSBP)

• keeps ssDNA from re annealing

• binding is cooperative

dna primase

synthesis primer which provides 3'OH to polymerase

primer is a short stretch of RNA

dna polymerase III

• DNA polymerase synthesizes most of DNA

• 3’-5’ exonuclease (proofreading)

• 5’-3’ polymerase activity

dna polymerase I

• removes primer (5' - 3' exonuclease)

• same functions as dna pol III

  • 3’-5’ exonuclease (proofreading)

  • 5’-3’ polymerase activity

describe dna replication of both the leading and lagging strand using the 7 enzymes

1. leading and lagging strands are separated by dna helicase at a replication origin (ori). two repliction forks are formed at one ori, but only one will be followed here:

2. rna primers are synthesized on both strands by dna primase.

3. on the leading strands, dna polymerase III begins replication by extending the 3' end from the primer.

4. lagging strand is not replicated bc dna poly III is unable to extend a 5' end.

5. more primers are added on lagging strand.

6. replication continues on the leading strand.

7. replication begins from a second primer on the lagging strand since dna poly III now has an available 3' end and a dna template (discontinuous replication) creating okazaki fragments

8. another primer is added to the lagging strand by primase

9. older primers are removed by dna poly I and newer primers are added where the dna is unwound

10. steps 5-9 are repeated

11. two copies of dna molecules are completed

*during replication, dna gyrase functions to release the strain on the molecule and ssbp keeps the ssdna fro reannealing, ligase seals okazaki fragments, and it is semiconservative replication*

dna ligase

seals okazaki fragments

• links nucleotides (okazaki fragments) together by forming phosphodiester bonds

plasmid vs episome

an episome is a plasmid that has the ability to integrate into the host chromosome. thus…

• all episomes are plasmids, not all plasmids are episomes

f+ cell

contains a complete f plasmid (donor)

f- cell

does not contain the f plasmid (acceptor)

f' cell

contains part of the f plasmid or an f plasmid with other bacterial genes (donor)

Hfr cell

contains the f plasmid integrated into bacterial genome (donor)

traA product

pili are hollow hair like projects on surface 2-3 micro meters long

tra S and tra T products

surface exclusion proteins prevents donor cell from mating with another donor cell

12 other tra products

needed for modification and assembly of pili and to stabilize initial association between donor and f- cells

tra M

recognizes a mating pair

pili / pilus

initiates conjugation, but disassembles as cells are brought into contact and transfer tube is formed

tra Y

binds near oriT and recruits traI

tra I

nicks oriT and forms a covalent bond with the 5' end of the nick

Agrobacterium

• tumor-forming plant pathogen

• function: tumor formation in plants from bacterial species

what is the Ti plasmid of agrobacterium

tumor inducting plasmid where the plant cell will receive foreign gene but does not becomes transformed into a tumor

virA

activitates virG by phosphorylation

virG-PO4

bids to virB, C, D, E, F promoters and activates them

virB

encodes a channel

virD

encode endonuclease and initiates nicking at correct locations of T-DNA

virE

encodes ssbp and binds to excised tdna to form "T-complex"

T-DNA

contains genes to generate transformed tumor state and synthesize opines

opines

synthesized by plant to give bacteria source of N and C compounds

(what the bacteria eat)

virF

product that is created and confers plant specificity

• disguises own DNA as plant matter

chv chvA, chvB, and pscA

chv chvA, chvB, and pscA codes for structures that are required for initial binding of bacteria to plant cell

Auxin

plant growth hormone

• reponsible for making tumors along w/ cytokinin

Cytokinin

Plant hormone for rapid cell division

• reponsible for making tumors along w/ auxin

Virulence Region

genes needed to inject T-DNA into plant cell

Transgenic plant & benefits

• Plants that have been genetically modified to express new or impoved phenotypes through the introduction or overexpression of genes

• BENEFITS:

  • increased resistance to insects

  • higher nutritional output and fruit yield

  • increased tolerance to environmental stressors (i.e., drought, heat, salinity, etc.)

Fidelity

how accurate the polymerase incorporates base pairs

DNA pol has ___ processitivity & ____ fidelity

high ; high

DNA polymerase alternates between what modes? draw models

• polymerizing mode (catalyzing DNA 5’-3’)

  • DNA wll lie across the palm in a groove formed by fingers and a thumb. this positioning allows the thumb to ratchet dna through the complex one base at a time as new nucleotides are added. the 3’ end of the growing chain is anchored by the fingers and palm. the dna is held into position by contacts between amino acids and the phosphodiester backbone that allows bases to be added that are complementary to the template

• editing mode (editing DNA 3’-5’)

  • As each new base is polymerized, it is checked for accuracy and fit. if the wrong base becomes incorporated, the strange conformation will not allow it to exit the polymerization site. instead, it is rotated into the exonuclease domain, and the incorrect base is removed by the 3’ to 5’ exonuclease activity. the DNA strand is then returned to the polymerizing site of the complex to try again.

plasmids:

circular extachromosomal entities in bacteria with accessory genes

F+/F- conjugation model

Hfr / F- model

Agrobacterium infection pathway model