BCH5413 TEST 2 QUESTIONS AND
ANSWERS ALL REVISED AND
UPDATED
Function of clamp loader
- ask whether loader is catalytic or loads one clamp per DNA and then stops - Answer--
circular ssDNA + primer + clamp/Pol subunit
- with increasing amount of clamp loader, increasing synthesis of DNA is seen
*result shows that it functions catalytically (one loader can load clamp onto 10
molecules of template)
Function of clamp loader
- ask whether loader remains associated with pre-initiation complex or whether it comes
off - Answer-- circular ssDNA template, plus radioactively labeled clamp, clamp loader,
single strand binding protein, ATP
- clamp remains in high MW, while loader remains in low MW
*means clamp stays on DNA but loader does not and is free
Does Pol 3 release and recycle? - Answer-- Pol3 + dNTP + M13 template w/clamp +
template w/o clamp
- results showed synthesis of DNA increased in the strands with a clamp
*after synthesis Pol3 is releasing and binding to another template if a clamp is available
Can Pol3 unload the beta clamp - Answer-- before Pol3, clamp is in large MW
consistent with them being bound
- after Pol3, clamp is in small MW consistent with free clamp from DNA molecule
If the beta clamp is unloaded, can it be recycled? - Answer-- in presence of clamp, Pol3
+ ATP .. with increase of clamp, increasing DNA synthesis
*results say 'yes'
What does the gamma complex do?
- experiment - Answer-unloading activity
- results showed large MW converting to small MW
,*demonstrates the gamma complex has clamp unloading activity
Termination of replication in E. coli - Answer-- there are multiple 'ter' sites
- they are binding sites for TUS proteins
- TUS-ter complex slow down and pause replication fork
*end up with two circular daughter chromosomal molecules that are interlocked (need to
be separated before cell division)
How are the two circular daughter chromosomal molecules separated? - Answer-
topoisomerase
- cleaves one and allows other to pass through
The problem of replicating ends of linear chromosomes in eukaryotes
- what is the solution - Answer-telomerase
- elongates the template 3' end of both strands
- allows synthesis of primer for 5' end (fills in gap generated by removal of previous
primer)
Activity and function of telomerase - Answer-- ends of linear chromosomes have short
repeat sequence
- RNA component of telomerase is homologous to repeat at ends of linear
chromosomes and base pairs at the 3' end
- telomerase shifts and has sequence that is synthesized to regenerate the end of the
linear strand (can occur multiple times)
- now that 3' end is elongated, primase can synthesize RNA primer homologous to
extended repeat synthesis
Mechanism for protecting ends of linear chromosome - Answer-telomere
- T and D loop
DNA replication and nucleosomes
- what happens to nucleosomes when replication fork approaches
*another theory - Answer-- nucleosome disassembles and H3/H4 tetramers remain on
DNA and randomly assort between leading and lagging strands
- presumably H3/H4 tetramers randomly assort between the two daughter strands of
DNA
- newly synthesized or old H2A/H2B dimers associate with the tetramers to reconstitute
chromatin structure on leading and lagging strands
*another theory suggests that H3/H4 tetramers separate to H3/H4 dimers and randomly
reassort with newly synthesized H3/H4 dimers + newly synthesized or old H2A/H2B to
reconstitute nucleosome structure
Overall steps behind replication fork and nucleosomes - Answer-disassemble
nucleosomes in front of replication fork and reassembling behind replication fork on two
daughter strands using old histones from parental DNA + newly synthesized to
reconstitute chromatin structure
,Methylation: a primitive immune system for bacteria
- hds-R - Answer-- plasmid transforms into cells are protected (restriction enzyme not
expressed)
Methylation: a primitive immune system for bacteria
- mcrA/mcrB/mrr - Answer-degrades foreign DNA that is not properly methylated
- can occur to DNA obtained from mouse/human cells containing CpG methylated DNA
(can interfere with cloning of mammalian genomic DNA)
pCR 2.1 - TOPO - Answer-Tac Pol adds A-overhangs to PCR products
TOPO cloning
- what it stands for - Answer-TOPO = topoisomerase
- contains lacZa (can use blue/white color screening)
- f1 Ori (can make ssDNA)
- amp-r
- pUC Ori
drawback: PCR product doesnt get directionally cloned
PCR cloning: test digest - Answer-- restriction enzyme site in pCR 2.1 TOPO + PCR
product
- 5' -> 3': will yield fragment of 300 and 4600 band
- 3' -> 5': 700 and 4200 band
Protein expression in E. coli - Answer-- clone DNA out of TOPO vector into downstream
plasmid capable of expressing protein
- expression plasmid encodes lacZ gene (IPTG in environment will induce protein
expression); will product beta-galactosidase protein
*can also be done by having a plasmid expression vector, lacZ gene to transformed E.
coli -> protein expressed
Shuttle vector: replication in E. coli and yeast - Answer-- cloning steps can be
performed in E. coli, protein expression takes place in eukaryotic cell (yeast)
yeast shuttle vector
- contains... - Answer-- polylinker
, - Ori
- amp-r
- CEN: ensures segregation
- ARS (ori to be copied within yeast)
- URA3 (uracil synthesis; selection)
Yeast shuttle vector: pESC-URA - Answer-URA3 for yeast selection required for growth
on media without uracil
Mammalian shuttle vector
- pcDNA3.1 - Answer-- contains f1 ori (ssDNA)
- amp-r
- pucOri (copying in E. coli)
- pCMV (promoter to express in mammalian cell)
- has epitope tag
- neomycin resistance (selectivity)
*promoter will always be active, does not need to be induced
cDNA library construction
- standard approach steps in isolating a cDNA clone - Answer-1. isolate mRNA
2. convert to ds cDNA
3. choose vector and insert cDNA
4. create phage or bacterial library
5. screen
6. verify identity
Making a cDNA Library - Answer-- isolate mRNA
- make cDNA
- perform second strand synthesis for ds cDNA and add C overhangs (sticky ends)
- clone into vector with complementary G overhangs
- anheal and ligate
Packaging cDNA library into bacteriophage lambda
- problems and solution - Answer-Problem:
- transformation of plasmids (DNA) into bacterial cells is inefficient
Solution
- use bacterial virus (phage) vector, not a plasmid
Packaging cDNA library into bacteriophage lambda
- steps - Answer-- mRNA transcribed into ds cDNA
- ligate ds linker to all ends of cDNA
- can be cleaved with EcoR1 for sticky ends
- complementary overhang vector added
- in vitro packaging to create recombinant virus particles
ANSWERS ALL REVISED AND
UPDATED
Function of clamp loader
- ask whether loader is catalytic or loads one clamp per DNA and then stops - Answer--
circular ssDNA + primer + clamp/Pol subunit
- with increasing amount of clamp loader, increasing synthesis of DNA is seen
*result shows that it functions catalytically (one loader can load clamp onto 10
molecules of template)
Function of clamp loader
- ask whether loader remains associated with pre-initiation complex or whether it comes
off - Answer-- circular ssDNA template, plus radioactively labeled clamp, clamp loader,
single strand binding protein, ATP
- clamp remains in high MW, while loader remains in low MW
*means clamp stays on DNA but loader does not and is free
Does Pol 3 release and recycle? - Answer-- Pol3 + dNTP + M13 template w/clamp +
template w/o clamp
- results showed synthesis of DNA increased in the strands with a clamp
*after synthesis Pol3 is releasing and binding to another template if a clamp is available
Can Pol3 unload the beta clamp - Answer-- before Pol3, clamp is in large MW
consistent with them being bound
- after Pol3, clamp is in small MW consistent with free clamp from DNA molecule
If the beta clamp is unloaded, can it be recycled? - Answer-- in presence of clamp, Pol3
+ ATP .. with increase of clamp, increasing DNA synthesis
*results say 'yes'
What does the gamma complex do?
- experiment - Answer-unloading activity
- results showed large MW converting to small MW
,*demonstrates the gamma complex has clamp unloading activity
Termination of replication in E. coli - Answer-- there are multiple 'ter' sites
- they are binding sites for TUS proteins
- TUS-ter complex slow down and pause replication fork
*end up with two circular daughter chromosomal molecules that are interlocked (need to
be separated before cell division)
How are the two circular daughter chromosomal molecules separated? - Answer-
topoisomerase
- cleaves one and allows other to pass through
The problem of replicating ends of linear chromosomes in eukaryotes
- what is the solution - Answer-telomerase
- elongates the template 3' end of both strands
- allows synthesis of primer for 5' end (fills in gap generated by removal of previous
primer)
Activity and function of telomerase - Answer-- ends of linear chromosomes have short
repeat sequence
- RNA component of telomerase is homologous to repeat at ends of linear
chromosomes and base pairs at the 3' end
- telomerase shifts and has sequence that is synthesized to regenerate the end of the
linear strand (can occur multiple times)
- now that 3' end is elongated, primase can synthesize RNA primer homologous to
extended repeat synthesis
Mechanism for protecting ends of linear chromosome - Answer-telomere
- T and D loop
DNA replication and nucleosomes
- what happens to nucleosomes when replication fork approaches
*another theory - Answer-- nucleosome disassembles and H3/H4 tetramers remain on
DNA and randomly assort between leading and lagging strands
- presumably H3/H4 tetramers randomly assort between the two daughter strands of
DNA
- newly synthesized or old H2A/H2B dimers associate with the tetramers to reconstitute
chromatin structure on leading and lagging strands
*another theory suggests that H3/H4 tetramers separate to H3/H4 dimers and randomly
reassort with newly synthesized H3/H4 dimers + newly synthesized or old H2A/H2B to
reconstitute nucleosome structure
Overall steps behind replication fork and nucleosomes - Answer-disassemble
nucleosomes in front of replication fork and reassembling behind replication fork on two
daughter strands using old histones from parental DNA + newly synthesized to
reconstitute chromatin structure
,Methylation: a primitive immune system for bacteria
- hds-R - Answer-- plasmid transforms into cells are protected (restriction enzyme not
expressed)
Methylation: a primitive immune system for bacteria
- mcrA/mcrB/mrr - Answer-degrades foreign DNA that is not properly methylated
- can occur to DNA obtained from mouse/human cells containing CpG methylated DNA
(can interfere with cloning of mammalian genomic DNA)
pCR 2.1 - TOPO - Answer-Tac Pol adds A-overhangs to PCR products
TOPO cloning
- what it stands for - Answer-TOPO = topoisomerase
- contains lacZa (can use blue/white color screening)
- f1 Ori (can make ssDNA)
- amp-r
- pUC Ori
drawback: PCR product doesnt get directionally cloned
PCR cloning: test digest - Answer-- restriction enzyme site in pCR 2.1 TOPO + PCR
product
- 5' -> 3': will yield fragment of 300 and 4600 band
- 3' -> 5': 700 and 4200 band
Protein expression in E. coli - Answer-- clone DNA out of TOPO vector into downstream
plasmid capable of expressing protein
- expression plasmid encodes lacZ gene (IPTG in environment will induce protein
expression); will product beta-galactosidase protein
*can also be done by having a plasmid expression vector, lacZ gene to transformed E.
coli -> protein expressed
Shuttle vector: replication in E. coli and yeast - Answer-- cloning steps can be
performed in E. coli, protein expression takes place in eukaryotic cell (yeast)
yeast shuttle vector
- contains... - Answer-- polylinker
, - Ori
- amp-r
- CEN: ensures segregation
- ARS (ori to be copied within yeast)
- URA3 (uracil synthesis; selection)
Yeast shuttle vector: pESC-URA - Answer-URA3 for yeast selection required for growth
on media without uracil
Mammalian shuttle vector
- pcDNA3.1 - Answer-- contains f1 ori (ssDNA)
- amp-r
- pucOri (copying in E. coli)
- pCMV (promoter to express in mammalian cell)
- has epitope tag
- neomycin resistance (selectivity)
*promoter will always be active, does not need to be induced
cDNA library construction
- standard approach steps in isolating a cDNA clone - Answer-1. isolate mRNA
2. convert to ds cDNA
3. choose vector and insert cDNA
4. create phage or bacterial library
5. screen
6. verify identity
Making a cDNA Library - Answer-- isolate mRNA
- make cDNA
- perform second strand synthesis for ds cDNA and add C overhangs (sticky ends)
- clone into vector with complementary G overhangs
- anheal and ligate
Packaging cDNA library into bacteriophage lambda
- problems and solution - Answer-Problem:
- transformation of plasmids (DNA) into bacterial cells is inefficient
Solution
- use bacterial virus (phage) vector, not a plasmid
Packaging cDNA library into bacteriophage lambda
- steps - Answer-- mRNA transcribed into ds cDNA
- ligate ds linker to all ends of cDNA
- can be cleaved with EcoR1 for sticky ends
- complementary overhang vector added
- in vitro packaging to create recombinant virus particles
