Sheet 11--Test 4 Flashcards
How did watson and crick propose that DNA is replicated?
semi conservative method–each “old” strand used as a template for the “new” strand
each DNA strand has one old and one new part that are the DNA strand
DNA polymerase–chooses nucleotides that have been preassembled and put them in order using “old” DNA as the pattern
How did meselson and Stahl prove that Watson and Crick were correct?
==labeled DNA with heavy nitrogen- 15^N
- parental DNA, both sides has heavy nitrogen
- -transferred to medium contain 14^N
- -Where does normal nitrogen end up?
- *paper
What does it mean to say DNA replication is semi conservative?
a newly synthesized DNa molecule consists of one “old” strand and one “new” strand
–semiconservative
You have identified a new virus with a single stranded DNA genome. After centrifuging DNa from infected cells in CsCl you obtain two bands with different density. One is 20% A, 30% C,
20% A 20% T 30%C 30% G
this is a single stand DNA becuases bases arent paired
–single stranded DNA has no complementary base pairing.
What are the problems which must be solved in order to replicate DNA?
Open it, keep it open, prime, copy, anneal (join segments of DNA together.
Why are there so many proteins in a DNA replication complex?
replication involves solving many problems, each problem is solved by a different protein. (enzyme)
How do cells know where to start replicating DNA?
begins at origin of replication
- -initiator proteins bind here and recruit replication complex (helicase, gyrase, RNA polymerase, DNA polymerase)
- -tell DNA polymerase where to start
what is an origin of replication?
specific sequence of DNA nucleotides where DNA is opened and untwisted by helicase; recognized by initiator proteins.
which proteins bind to the origin or replication?
Initiator proteins– recognize and bind to origin of replication/ recruit replication complex (ex. helicase,gyrase)
–forms a complex that opens the helix and separates the strand
Specifically:
–bacteria use initiator proteins
–Eukaryotes use ORC: origin recognition complexes
these proteins bind to specific DA sequences to begin replication
what does helicase do?
unwinds DNA
What does Gyrase do, and why is necessary?
relieves supercoilng because the unwinding of DNA helix produces torsional strain.
What do SSB proteins do, and why are they necessary?
- Single Strand bind protein
- -stabilize single stranded regions
Why do cells need an RNA primer to start DNA replication?
- -DNA polymerase can only add nucleotides to an existing molecule
- -DNA polymerase adds DNA nucleotides to an RNA primer
- -add to 3’ side
what is the role of the sliding clamp?
subunit of DNA polymerase 3 (enzyme that adds DNA nucleotides makeing “new strand”)
–forms a ring around DNA template, holding the enzyme in place
how does the cell determine which base to add next?
only one base fits
–use complementary strand as template
What holds the new base in place until DNA polymerse has catalyzed the phosphodiester bond?
hydrogen bonds between complementary bases.
where does the energy needed to catalyze the phosphodiester bond come from?
Hydroylsis of two PO4 molecules
–use ATP
ATP –> ADP +P + energy
What is DNA proof reading and how does it work?
- -only correct DNA can exit replication process
- -proof reading is the most important aspect of replicating DNA correctly
- -if bass are mis-paired, process backs up and tries again
- -most DNA mistakes are corrected this way
why is there an RNA polymerase in every replisome?
RNA polymerase is called primase
–makes a short piece or RNA called a primer for DNA polymerase to add to.
why is there a ribonuclease in every replisome?
ribonuclease removes RNA primer
–will be replaced with DNA nucleotides
why do we say that DNA replication is semi discontinuous?
leading strand is copied continuously towards replication fork
–agging strand is made in fragments away from replication fork
why are there leading and lagging strands at every DNA replication fork?
DNA polymerase can only add in 5’ to 3’ direction; antiparallel strands are copied by different mechanisms
What is an Okazaki fragment?
short pieces of DNA with a short stretch of RNA at each 5’ end are found temporarily on the lagging end.
why is DNA ligase associated with each DNA replication complex?
attaches anneal Okazaki framents together by creting phosphodiester bonds
what is the loop model for replicating DNA
- -DNA is fed through replisome
- -lagging stand is looped out
- -both DNA polymerase move in the same direction
- -see diagram in text**
why is the loop model necessary?
both leading and lagging strands are replicated in same direction
why do eukaryotic cells have multiple origins of replication on every chromosome?
because chromosomes are large and there is a lot of DNA. One origin of replication would take too much time to replicate.
what is an ARS?
Autonomously Replicating Sequences
- -section of DNA replicated at each origin of replication
- -average 100,000 base pairs in length
- -each zone replicated as a unit
- -increases speed of replication
What is an ORC?
Origin Recognition complex
- -binds to ARS element
- -make sure, DNA is only replicated once by recruiting licensing factors.
How do Eukaryotic cells ensure that each origin of replication only fires once per cell cycle?
- -each ARS is replicated as a discrete unit
- -licensing factors ensure ARS is replicated once per S phase; these fall off (as do activation factors) once replication starts + are thrown out of nucleus they dont reatach until after mitosis (late telophase)
What are telomers?
- -DNA sequence at the ends of Eukaryotes chromosommes
- -in humans, consist of 250-7000 repeats of CCCTAA
- -protect the ends of chromosomes since special proteins bind the telomeres
- -with each replication, a telomere is removed.
why is replicating the ends of linear DNA molecules a problem?
replication of chromosomes ends is a problem:
- -directionality of polymerases plus need for a primer are the problems
- -no problem with leading strand, but lagging strand only replicated to last primer; leading strand is therefore longer than lagging strand
- -each mitosis gets 200 base pairs shorter
what is telomerase, and what does it do?
Telomerase–enzyme that replcaes missing bases
- -adds nucleotides to telomeres
- -uses an internal RNa as a template (not the DNA itself)
- -use of internal RNA allows short segments of DNA to be made
- -these segments are repeated nucleotide sequences complementary to the RNA of the enzyme; other strand is completed by normal DNA synthesis.
What is the telomerse theory of aging?
Whithout the action of telomerase, chromosome ends shorten
- -normal cells undergo a specified number of cell divisions correlated with length of telomere
- -telomerase is active in embryonic cells and cells of children, but low in adult somatic cells (except cells like lymphocytes which divide as part of their function)
- -there is a relationship between cell senescence (aging) and telomere length; mature cells dont make telomerse.
what does telomerase have to do with cancer?
cancer cells show activation of telomerase, which allows chromosomes to maintain their length. This is one aspect of cancer that allows for cell division that continues indefinitly.
–telomerase is a symptom, not a cause, of cancer/ Serum telomerase is used to diagnose cancer.
What is a mutagen?
agents (radiation chemicals) that damage DNa.
DNA sequence is incorrect (A,C,T,G)
How does UV damage DNA, and how is this damage repaired?
Uv causes formations of thymine \+ pyrimidine dimers -adjacent thymine bases link together T T A A becomes T-T and A-A
what is photolyase?
Photo repair occurs: enzyme called photolyase absorbs visible light and uses the energy to cleave the thymine dimer.
- -sunlight in UV range causes damage
- -sunlight in visible range used to repair damage
what is nucleotide excision repair?
- Damaged region of DNA is removed and then replaced by DNA synthesis
- -involves:
1) recognition of damage
2) removal of damaged region
3) resynthesis of removed segment using undamaged strand as template
What causes Xeroderma pigmentosum?
- -disorder resulting from inability of cells to correct DNA damage due to UV
- -8 genes are involved in DNA repair caused by UV
- -individual is ultra sensitive to UV
what is base excision repair?
repair places altered or missing bases
what is mismatch repair?
fix new DNA to match old DNA
what is the cause of Hereditary Nonpolyposis colorectal cancer?
bad mismatch repair
How do X-rays damage DNA?
x rays damage DNA by causing double strand breaks
why do defective BRCA1 or BRCA2 genes increase risk for cancer?
–BRCA1 and BRCA2 work with protein RAD51 to repair double stand breaks. If these are defective, DNA damage wont be corrected
- -If both brac1 are defective 80% of cancer risk
- -if brac2 are defective 70% of cancer risk
Why did Garrod conclude that some disease have genetic basis?
disease was ‘in the family’ inherited
what evidence supports the hypothesis that each gene encodes a protein?
destroy gene, no enzyme [ a gene contains instructoins for the production of a protein]
–product of a gene is a protein
how did beadle and tatum show that genes are instructions for making enzymes?
- one gene one enzyme hypotheis
- deliberately destroyed genes, then looked for heritable defects in enzymes
- -mutants unable to grow on minimal media
- -identified missing nutrient by adding specific chemicals, one at a time
- -each allele coded for a different enzyme in pathways used for orginine production
genes store information for the production of proteins. information is stored in the sequence of nucleotides of the gene.
what is a plasmid and what does every plasmid have?
.All plasmids contain origins of replication which tell the cell how to replicated the plasmid and how many copies to make
What did the Hershey-chase (waring blender) experiement prove?
DNA is the hereditary matierial
Why has PCR been so useful for molecular biologists?
it allows you to make enough DNA to analyze starting from very small samples
What did Meselson and Stahl conclude that DNA replication is semi conservative?
after switching cells grown on N15 for many generations to N14, all of the F1 generation of DNA molecules were of intermediate density
Why did you add calcium chloride to your bacteria and why did you heat shock it?
Calcium chloride loosens up cell wall so plasmid can get through
heat shock allows plasmid to center cytoplasm
Why did you spread your bacteria on plates containing ampicillin?
cells which took up plasmid should be able to grow on medium containing ampicillin; bacteria that did not take up plasmid will be killed by ampicillian
–this is how transformants are selected.
what is a restriction enzyme and what is their purpose in nature?
restriction enzymes cut DNA at specific sequences called restriction. They are produced by bacteria as a defense against viruses
how do bacteria protect their DNA from being destroyed by their own restriction enzymes?
Bacterial cells have a restrictino endonuclease/methylase pair that protects bacterial recognition sequences
–bacterial DNA is methylated which protects it from restriction
restriction mapping
determine the relative positions of restriction sites
55 fingerprint human DNA
we use PCR to detect which version we have for each of the 13 CODIS mictosatellite loci used by the FBI
-chromosome #16 for alu sequence
-chromosome #4 for microsattellites
cut dNA with restirction enzymes run dNA on gel
DNA separates based on size creates a dNA fingerprint
What binds at the origin of replication?
initiator proteins–these tell DNA polymerase where to start.
What is the replication complex?
studd used for replication–helicase, gyrase, DNA polymerse where to start
What is the replication complex?
stuff used for replication–helicase, gyrase, DNA polymerse
In eukaryotic cells, what binds to the origin of replication?
ORC– Origin recognition complexes, these proteins bind to speficic DNA sequences to begin replication
In prokaryotic cells, what binds to the origin of replication?
Initiator proteins
What is the most important aspect of replicating DNA?
initiator proteins
How are most DNA mistakes fixed?
by DNA proofreading
what is an RNA primer?
short piece of RNA made by primase
how is leading strand of DNA made
in a continuous fashion
how is lagging strand made
in fragments away from the replication fork
what does it mean that DNA strands are antiparallel
one strand runs 3’ to 5’ and the other strand runs 5’ to 3’
what s a section of DNA replicated at each origin of replication?
ARS–autonomously replicating sequences
how long is the average ARS?
Autonomously replicating sequence
100,000 base pairs
What is the function of the ARS? autonomusly replicating sequence
increases speed of replication because each zone is replicated as a unit
what binds to the ARS element?
ORC–origin recognition complex
What is the function of the ORC–origin recognition complex?
that DNA is only replicated once by recruiting licensing factors
what happens to ARS after S phase?
they fall off, along with activation factors once replication starts and are thrown out of nuclues so the dont reattach until after telophase.
What is the enzyme that replaces missing bases?
telomerse
what is the template of telomerse?
internal RNA, this allows for short segments of DNA to be made
when is telomerse active?
in embryonic cells and cells of children…also cancer
what is the number of cell division correlated with?
length of telomere
what is a licensing factor
a protein that allows for an origin of replication to begin DNA replication at that site
what enzyme absorbs visible light in order to cleave thymine dimer?
photolyse
wat is the result of Xeroderma pigmentosum?
sensitivity to UV
what types of cells have plasmids?
prokaryotic, bacteria
what is produced by bacteria as a defense against viruses?
restriction enzymes
what bonds connect okazaki fragments
phosphodiester bonds
how are transformants selected?
by spreading bacteria on plates containing ampicilian
