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Cancer arises from cells that have lost the capacity to ____ and _____ in a controlled manner as a consequence of damage to _____ that encode _____ that govern the ____ ____
- grow and divide
- cell cycle
If genetic material was flawlessly perpetuated, genetic variation needed to drive ________ would be lacking and ___ ____ would not have arisen
Two important sources of mutations
- inaccuracy in DNA replication
- chemical damage to the genetic material
Replication errors arise from _________, which imposes an upper limit on the accuracy of ____ _____ during DNA replication.
- base pairing
Errors in replication and damage to DNA have two consequences
- permanent changes to the DNA aka mutations
- some chemical alterations to DNA prevent its use as a template for replication and transcription
The effects of mutations generally become manifest only in the progeny of the cell in which the _______ ______ occurred, but DNA lesions or ______ changes to the DNA that impede _______ or _______ can have immediate effects on cell _______ & ______
- sequence alteration
- replication or transcription
- function & survival
What must the cell do when lesions occur
- it must scan the genome to detect errors in synthesis and damage to the DNA
- it must then mend the lesions and do so in a way that, if possible, restores the original DNA sequence
The simplest mutations are switches of one _____ for another. What are the two main types?
mutation where we have pyrimidine to pyrimidine and purine to purine substitutions, such as T to C and A to G
mutations were we have pyrimidine to purine and purine to pyrimidine substitutions such as T to G or A and A to C or T
Other simple mutations involve _____ or _____ of nucleotides or a small number of nucleotides. Mutations that alter a single nucleotide are called ______ _______
- insertions or deletions
- point mutations
Other kinds of mutations cause more drastic changes in DNA, such as extensive ______ and ______ and gross rearrangements of _______ structure.
The more drastic changes/mutations might be caused, by the insertion of a ______, which typically places ______ of nucleotides of foreign DNA in the ______ or ______ sequences of a gene or by the aberrant actions of cellular _________ processes
- coding or regulatory
The overall rate at which new mutations arise spontaneously at any given site on the chromosome ranges from ~10-6 to 10-11 per round of ____ _______ with some sites on the chromosome being ____ ______, where mutations arise at high frequency, and other sites undergoing alterations at a comparatively _____ ______.
- DNA replication
- hot spots
- lower frequency
One kind of sequence that is particularly prone to mutation merits special comment because of its importance in human genetics and disease. These _____-_____ sequences are repeats of simple di, tri, or tetra-nucleotide sequences known as ____ ________
- mutation prone
- DNA microsatellite
One well known example of DNA microsatellite involves repeats of the _______ sequence CA, where stretches of it are found at many widely scattered sites in the chromosomes of humans and some other _______. The replication machinery has difficulty copying such repeats accurately, frequently undergoing ______.
Slippage causes _______ or _______ in the number of copies of the ______ sequences. As a result, the CA repeat length at a particular site on the chromosome is often highly ________ in the population. This provides a convenient physical marker for mapping inherited ______ (such as?)
- increases or decreases
- mutations that increase the propensity of certain diseases in humans
The proofreading exonuclease is not foolproof, some misincorporated nucleotides escape detection and become a mismatch between the _____ ______ strand and the ______ strand.
- newly synthesized strand
- template strand
_____ different nucleotides can be misincorporated opposite of each the four kinds of nucleotides in the template strand for a total of _____ possible mismatches. If the misincorporated nuclotide is not subsequently ______ and _____, the sequence change will become permanent in the genome
- detected and replaced
How do misincorporated nucleotides become permanent after escaping detection and replacement
- During a second round of replication, the misincorporated nucleotide, will now be part of the template strand
- It will direct the incorproation of its complementary nucleotide into the newly synthesized strand and the mismatch will no longer exist.
- Instead, it will have resulted in a permanent change (mutation) in the DNA sequence
Mismatch repair system is a mechanism for _______ and ______ mismatches. It increases the accuracy of DNA synthesis by an additional _____ or _____ orders of magnitude.
- detecting & repairing
- two or three
Two problems the mismatch repair system faces
- it must rapidly scan the genome for mismatches which are transient or eliminated following a second round of replication when they result in mutations
- the system must correct the mismatch accurately; that is, it must replace the misincorporated nucleotide in the newly synethesized strand and not the correct nucleotide in the parental strand
In e. coli, mismatches are detected by a ______ of the mismatch repair protein ______. _____ scans the DNA, recognizing mismatches from the distortion they cause in the DNA ______.
MutS embraces the mismatch-containing DNA, inducing a pronounced kink in the DNA and a _______ change in MutS itself
A key to the specificity of MutS is that DNA containing a mismatch is much more readily _______ than properly base paired DNA. MutS has an ______ activity that is required for mismatch repair, but its precise role in ______ is not understood.
The complex of MutS and the mismatch-containing DNA recruits _____, a second _____ component of the repair system. It in turn activates _____
an enzyme that causes an incision or nick on one strand near the site of the mismatch.
Nicking is followed by the action of a specific ________(UvrD) and one of three _______. The ______ unwinds the DNA, starting from the ______ and moving in the direction of the site of the ________, and the ________ progressively digests the displaced single strand, extending to and beyond the site of the _______ nucleotide
The exonuclease progressively digesting the displaced single strand produces a _____ _____ gap, which is then filled in by _____ _____ ______ and sealed with ____ _____. The overall effect is to _____ the mismatch and _______ it with the correctly base-paired nucleotide.
- single strand gap
- DNA pol III
- DNA ligase
How does the E. coli mismatch repair system know which of the two mismatched nucleotides to replace?
E. coli tags the parental strand by transient hemimethylation
The e. coli enzyme ____ ______ methylates A residues on both strands of the sequence _______. The sequence is widely distributed along the entire ______ (occurring once every 256 bp) and all of these sites are methylated by ____ _______.
- Dam methylase
- Dam methylase
When a rep fork passes through DNA that is methylated at _____ sites on both strands (_____ methylated), the resulting daughter DNA duplexes will be ________ (methylated on only the ______ strand). So, for a few minutes, until the _____ ______ catches up and methylates the newly synthesized strand, daughter DNA duplexes will be methylated only on the strand that served as a _______. Thus the newly synthesized strand is marked (it lacks a _____ group) and can be _______ as the strand for repair.
- fully methylated
- parental strand
- Dam methylase
The MutH proteins binds at _______ sites, but its _______ activity is normally latent. Only when MutH is contacted by ______ and _____ located at a nearby ______ (usually within a few hundred base pairs) can MutH become activated
- hemimethylated sites
- MutS & MutL
What does MutH do when actived
selectively nicks the unmethylated strand, thus only newly synthesized DNA in the vicinity of the mismatch is removed and replaced.
Methylation is referred to as a _____ device that enables the E. coli repair system to retrieve the correct sequence from the ______ strand if an error has been made during ______
Different _______ are used to remove ssDNA between the nick created by MutH and the mismatch depending on if the nick was made on the ____ or the ____ end of the DNA
Eukaryotic cells also repair mismatches and do so using homologs to ______ (called ____) and _____ (called ____ & ____)
- MSH or MutS homolog
- MLH & PMS
Even though euk cells have mismatch repair systems, the lack _____ and the clever trick of using ______ to tag the parental strand as found in E. coli. However, most bacteria are unable to use _______ and lack ____ _____
- Dam methylase
How does the mismatch repair system know which of the two strands to correct?
______ strand synthesis takes place discontinuously with the formation of ______ ________ that are joined to previously synthesized DNA by ____ _____.
- Okazaki fragments
- DNA ligase
Before the ______ step, the Okazaki fragment is separated from the previously synthesized DNA by a ____, which can be thought of as being the same as the _____ in E.coli by _____ on the newly synthesized strand.
- ligation step
Extracts of euk. cells will repair mismatches in artificial templates that contain a _____ and do so selectively on the strand that carries the _____. Recent results indicate that human homologs of ______ interact with the sliding clamp component of the replisome
Mutations arise not only from errors in replication, but also from _____ to DNA. Some is caused by environmental factors such as _______ and so called _____ which are chemical agents that increase ______ _____
- mutation frequency
DNA also undergoes spontaneous damage from ________. Ironic because the proper structure of the double helix depends on an _______ environment
Most frequent and important kind of hydrolytic damage is _______ of the base ______. Under normal physiological conditions, it occurs and generates an unnatural base in DNA called ______. The base then pairs with ______ and thus introduces that base in the opposite strand upon replication rather than the G that would have been directed by C.
_______ & ______ are also subject to deamination. It converts adenine to _______, which forms hydrogen bonds to _______ instead of _______. Guanine is converted to _______, which continues to pair with _______, although with _____ hydrogen bonds.
- Adenine & Guanine
Why does DNA have uracil instead of thymine?
If DNA naturally contained uracil instead of thymine, then deamination of cytosine would generate a natural base, which the repair systems could not recognize easily
The hazard of having deamination generate a naturally occurring base is illustrated by the problem caused by the presence of _________. Vertebrate DNA frequently contains _________ in place of cytosine as a result of the action of __________
Deamination of 5-methylcytosine generates ________, which obviously will not be recognized as an abnormal base and following a round of DNA rep. can become fixed as a ______ ________. Methylated Cs are _____ _____ for spontaneous mutations in vertebrate DNA
- C-T transition
- hot spots
DNA is damaged by ______, _______ and _______
Alkylation, oxidation and radiation
In alkylation, _____ or _____ groups are transferred to reactive sites on the bases and to _______ in the DNA backbone
Alkylating chemicals include_______ and the very potent laboratory mutagen N-methyl-N1-nitro-N-________. Where is one of the most vulnerable sites of alkylation.
The product of this methylation, ________, often often mispairs with ________ resulting in the change of G:C base pair into an A;T base pair when the _______ DNA is replicated
- keto group at carbon atom 6 of guanine
DNA is also subject to attack from reactive _____ species. These potent ______ agents are generated by _______ radiation and by chemical agents that generate ______ ______
- oxygen species (O2, H2O2, OH)
- oxidizing agents
- ionizing radiation
- free radicals
Oxidation of guanine for example, generates _____ (7,8-dihydro-8-oxoguanine). The product's adduct is highly ______ because it can base pair with ______ as well as with _______.
What happens when the oxoG adduct base pairs with adenine during replication
It gives rise to a G:C to T:A transversion, which is one of the most common mutation found in human cancers. Thus, perhaps the carcinogenic effects of ionizing radiation and oixidizing agents are partly caused by free radicals that convert guanine to oxoG
Yet another type of damage to bases is caused by ______ light. Radiation of approx. 260nm is strongly absorbed by the ______, one consequence of which is the photochemical fusion of two _______ that occupy adjacent positions on the same _______ chain
- polynucleotide chain
If the case is the photochemical fusion of two thymines, the fusion is called a _____ _______, which comprises a _______ ring generated by links between the carbon atom ___ & _____ of adjacent ______.
- thymine dimer
- 5 & 6
In the case of a thymine adjacent to a cytosine, resulting fusion is a ______-______ adduct
Ionizing radiation like _____ & _____ are particularly hazardous because they cause double strand breaks in the DNA, which are difficult to repair. If left unrepaired, double-strand breaks can be _____ to a cell.
- γ-radiation and X-rays
Ionizing radiation can directly attack (_____) the _______ in DNA's backbone. Alternatively, this radiation can exert its effect indirectly by generating reactive ______ species, which in turn, react with the ________ subunits..
Because cells require intact chromosomes to replicate their DNA, ionizing radiation is used therapeutically to kill rapidly _______ cells in ______ treatment. Certain anticancer drugs, such as bleomycin, also cause _____ in DNA. Ionizing radiation and agents like bleomycin that cause DNA to ______ are said to be clastogenic