Mutation rates are extremely low -
only ~_ nucleotide change per ___ nucleotides each time DNA is replicated.
There are _ proofreading mechanisms in DNA replication. Name them.
1) DNA polymerase checks the exact base pair geometry before it catalyzes the addition of a nucleotide to the growing DNA chain.
2) Exonucleolytic Proofreading provided by another catalytic site on the DNA polymerase called a 3'-to-5' proofreading exonuclease. SELF-CORRECTING enzyme
3) Stand-directed mismatch repair; detects distortion in the double helix caused by mismatches base pairs
-recognizes which is the template strand to remove a mismatched nucleotide
a new ___ primer synthesized by DNA primase is added to prime the DNA for replication in the __ to __ direction. The only primer is eventually replaced by _____ polymerase in _____ species. However in Eukaryotic cells a special ____ ____ enzyme replaces the old ____
RNA: 5'-3' direction; DNA repair enzyme; RNA
To seperate DNA strands the enzyme DNA ____ binds to the DNA to split it using ____ _____ as an energy source to break the hydrogen bonds.
Helicase; ATP hydrolysis.
____ straighten the region on DNA chain for DNA polymerase to synthesize. If they were absent the DNA template woulf form short base-paired _____
SSB single strand binding proteins ; hairpins. Makes it difficult for DNA polymerase to slide down the template smoothly.
How does the DNA polymerase remain attached to the template strand?
A sliding clamp + clamp loader utilize energy (ATP) to load and clamp DNA polymerase to the template for synthesis . The clamp loader dissociates as the clamp + polymerase synthesize in the 5'-3' direction.
In Bacteria, 2 enzymes ; DNA primase and DNA helicase link to form _____
This enzyme releases torsional strain from unwinding DNA. Describe how it works. Where does it bind?
Topoisomerase. Covelently attaches to DAN phosphate, breaking a phosphodiester linkage in one DNA strand. It binds downstream from the replication fork.
When topoisomerase breaks a phosphodiester bond to relieve torsion tension, how is the DNA rebonded?
The original phosphodiester bond energy is stored in the phosphotyrosine linkage, making the reaction reversible.
spontaneous re-formation of the phosphodiester bond regenerates both DNA helix and the DNA topoisomerase.
On the DNA strand, a exposed _____ group is found on the 5' end while an exposed ___ group is found on the 3' end.
Phosphate on 5' end and OH group on the 3`end
A doubled-ring nucleotide base consiting of Adenosine and Guanine
A single-ring nucleotide base consisting of cytosine, uracil, and thymine
Okazaki fragments are ___ np long in prokaryotes, and ____ long in eukaryotes
1000-2000 np long in prokaryotes
100-200 np long in eukaryotes
True/false: DNA synthesis is driven by a large favourable energy-free change
True. Caused by the release of pyrophosphate via hydrolysis of A-triphosphate
the ____ nucleotide has greater ____ for DNA polymerase then does the ______ nucleotide; makes the correct pairing more energetically favourable
correct; affinity; incorrect
____ does not need a self-correcting mechanism
the catalytic site for exonucleolytic removal is in the ___ of DNA polymerase
exonucleolytic proofreading is in the ___ to ___ direction
___ ____ synthesizes RNA primers on the lagging strand
An enzyme that joins the 3' end of the newer (formerly RNA primer) DNA fragment to the 5' end of the previous DNA fragment
DNA ligase catalyzes a covalent link
An advantage to using a RNA primer of DNA is;
: the ribonucleotides in the primer
automatically mark these sequences as “suspect copy” to be efficiently removed
and replaced. A self-correcting enzyme cannot start synthesis of chains de novo. Any enzyme that primes will of
necessity make a relatively inaccurate copy. Therefore mutation rates would be
In eukaryotes, the RNA primer is erased by _____ _. The gap is filled by ____ and covalently joined by ___
RNase H; DNA polymerase; DNA ligase.
Okazaki fragments are 100-200 np's long in eukaryotes
DNA helicase can only unwind in the 5' to 3' direction?
False. DNA helicase can unwind 5' to 3' as well as 3' to 5'
what two proteins are needed to open up the double-helix?
DNA helicase and SSB proteins
In regards to DNA helicase, ATP hydrolysis causes?
a shape change to propel itself rapidly alonf a single strand of DNA
SSB proteins bind to? Do they cover the whole DNA strand molecule?
Bind to the phosphate back bone and leave the bases exposed. The straighten the strand preventing formation of short hair pin helices
Another word for helix-destablilizing proteins?
Single-stranded binding proteins
True/false: On their own DNA polymerase quickly synthesizes DNA from the DNA template strand.
False. On their own DNA polymerase woulf quickly dissociate. An accessory protein, sliding ring clamp, keeps DNA polymerase firmly help to the template strand but dissociates when DNA polymerase encounters a double-stranded region
In reality most of the proteins are held together in a large and orderly ________ complex that rapidly synthesizes DNA
The close association of proteins of the multienzyme complex at the replication fork _____ efficiency.
Increases. This is made possible by the folding back of the lagging strand. Also, the clamp loader and the lagging strand DNA polymerase are kept in place as part of this protein machine even when they dissociate from the strand
Strand-directed mismatch detects _____
distortion in the DNA from misfit noncomplimentary base pairs. Recognizes the template and removes the newly synthesized nucleotide.
Describe the 3-step process of strand-directed mismatch repair
1) recognition of mismatch
2) excision of DNA segment containing the mismatch from the newly synthesized strand
3) resynthesis of the excised base using the old strand as a template
Strand directed mismatch repair system reduces the number of errors made in DNA synthesis by a factor off ___
2 proteins involved in strand-directed mismatch repar. What is their role?
MutS - binds specifically to the mismatch
MutL - scans nearby DNA for a nick. Once the nick is found, it triggers degradation of the nick strand all the way back to the mismatch
Where does topoisomerase I bind on the DNA strand?
to the phosphate back bone
Topoisomerase I requires ATP hydrolysis?
False, the covalent linkage that joins the DNA topoisomerase protein to the DNA phosphate retains the energy of the cleaved phosphodiester bond, amking resealing rapid without energy input. Unlike DNA ligase of which joining requires ATP hydrolysis.
3 steps topoisomerase II releases a intertwined doubled strand. What is the energy source to drive the reaction?
1) it breaks one double helix reversibly to create a DNA gate
2) it causes the second nearby double helix to pass through this break
3) it reseals the break and dissociates from DNA
ATP hydrolysis is used as an energy source.
Replication of DNA begins with ____ _____ _____
special initiator proteins
Bacterial chromosomes typically have __ origin of replication
there are always _ replication fork(s).
2 replication forks.
In prokaryotes, describe the method to govern the amount of replication.
The original OR is double methylated. As the OR is seperated and a new strand is synthesized, the double helix is now only hemi-methylated. (A-nucleotides are methylated). Hemi-methylated regions are resistant to replication. The OR is bound by an inhibitor protein, blocking access of the origin to initiator proteins.
This enzyme methylates OR's.
DNA methylase enzyme
In eukaryotes, replication occurs only during which phase of the cycle?
S phase - synthesis phase
Heterochromatin is ____ found in the ___ ___ phase.
condensed; late S phase.
Euchromatin is ___ _____ and is found in ____ S pahse
less condensed; early S phase
Telomeres consists of tandem repeats rich in _ nucleotides located at the ___ end
G nucleotides; 3'
this enzyme is similar to reverse transcriptase
telomerase consists of a ___ template and ___ that acts as reverse transcriptase
telomeres function to prevent a ___ of DNA
This protein is associated with telomeres and helps protect the end of chromosomes
Shelterin - found in association in the t-loop on the telomere
chromosomal shortening of telomere sequences to the point where coding region
is affected. Exceptions include?
Replicative cell senescence (aging); exceptions include cancer cells- where genes that code for telomerase become activated
Hydrolytic attack , oxidative damage, and uncontrolled methylation are examples of?
spontaneous DNA alterations that lead to DNA repair
______ is the most common change; releases
a guanine, as well as adenine from DNA by hydrolytic attack
This alteration converts a cytosine to an altered base Uracil
a covalent bond forms between 2 pyrimidines to
form a _____. The common cause is UV damage in skin cells. The
most common ?
pyrimadine dimer. The most common is a thymine dimer
2 types of accidental strand breaks can occur. Name them
Non-homologous double strand break: often involves short deletions or insertions that result in a loss of DNA
Homologous recombination: use the sister chromatid as a template to restore the double-strand break. More difficult to accomplish
Genetic recombination occurs through 3 mechanisms:
1) Homologous recombination : meiosis
3)Conservative site-specific recombination
Mechanisms 2+3 use mobile genetic elements to translocate DNA
What is a mobile genetic element?
Elements used in genetic recombination from mechanisms like transpositon and conservative site-specific recombination. They are moved from one location to another location within a genome or to another genome. To another genome includes virsuses ect.
A transposon moves ____ a genome
encoded by the transposon, an enzyme called ____ moves the transposon
transposase can move a transposon via 2 pathways. Name them
cut-and-paste pathways and replicative pathways
Non-retro retroviral transposons utilize what enzyme(s)?
Reverse transcriptase and endonucleases. Moves via RNA intermediate
a tranposon is how long?
1,000 to 12, 000 np's
True/false: Cut-and-paste mechanisms of a transposon can alter DNA causing a mutation.
True. The donor chromosome is identified through inverted repeat DNA sequences. The repeat sequences are brought together to form a loop and excised. The staggered breaks are repaired via DNA polymerase and DNA ligase. As a result, the insertion site is marked by short repeat sequences of the target DNA. Although the break from the donor chromosome is repaired, the process alters the DNA sequence, causing a mutation at the original site of the excised transposable element.
Describe the mechanism of replicated transposons.
DNA-only transposon. Transposon DNA is replicated from the original site and is inserted to the new site. The original transposon remains at the original site. Similar to cut-and-paste mechanisms.
True/false: Some transposons can move in both replicative and cut-and-paste mechanisms.
Cut-and-paste transposons can move only with additional energy input?
False. This mechanism can move without the input of external energy. The reaction begins and ends with the same number of phosphodiester bonds, it can occur without the additional input of energy
DNA-only transposons are so named because?
They only involve the movement of DNA. Predominant in bacteria
Cut-and-paste transposons leaves behind a hole in the chromosome. 3 situations can occur to fill this hole. Describe them
1) can be healed via homologous repair provided the chromosome has just been replicated and an identical copy of the damaged host sequence is available; restores the transposon to the original situation
2) in a diploid organism, the damage can be recombinationally repaired using the chromosome homolog - transposon will NOT be restored
3) Non-homologous end joining reaction can reseal the break; producing a mutiation at the site where the transposon was excised
DNA polymerase I is found in _____ cells and functions to;
removes and replaces RNA primers (also in excision repair of damaged DNA)
DNA polymerase ___ is the main polymerase for synthesis on the leading strand, as well as Okazaki fragments on the lagging strand.
This enzyme is found in _____ cell types and functions to make RNA oligonucleotides that are primers for DNA synthesis
both cells ; DNA primase
Initiator proteins are found in _____ cells and function to;
bind to the replication origin and initiate unwinding of DNA double helix
Telomerase is found in _____ cell types. It functions to;
using an RNA template, its protein part synthesizes DNA for extension of telomeres causing a 3' overhang that will be converted to double-stranded DNA
Another name for transposons that move within a genome
Viruses consist of nucleoc acid surrounded by a ____
capsid (protein coat)
A virus that infects bacteria
Retroviral-like retrotransposons require these enzymes for movement and move via which molecule?
Reverse transcriptase and integrase (transposase)
moves via RNA intermediate produced by the promoter in LTR ( long terminal repeats) at each end
If a virus does not have a ____ it will not leave the host cell
Jumping gene exist/extinct in human genes
extinct. They no longer exist
Nonretroviral retrotransposons use a protein complex composed of?
reverse transcriptase and endonuclease
In nonretroviral retrotransposons; RNA is attacked on the ___ end of the hnRNA.
3' poly-AAAA tail
Viruses enter the cell, then exit via ____ of the host cell
A viroid is?
small circular ssRNA molecule
no protein coat present
use RNA polymerase II to replicateinfect cetrain plant types
A plasmid is typically circular _____ molecule that self-replicates in the cytoplasm of many ______ and the _____ of some eukaryotic species cells.