DNA mutations and repair

Gene expression relies on underlying DNA sequence

mutation: a change in the DNA sequence that is passed to

descendants (cells and/or organisms, aka an inherited change

• point mutations, deletion, insertion

• some mutations may be beneficial

• mutation drives evolution of species

Types of mutations

location: somatic or germline cells ( gametes)

molecular change: substiution, frameshift, expanding nucleotide repeats

function: missense, nonsense, (silent, neutral, loss or gain of function)

Types of substiutions’s

Types of substitutions

• Transition: same type of nitrogenous base

• Transversion: switch to other type of nitrogenous base

• Frameshifts aka indels

• Expanding nucleotide repeats occur when a set of repeated nucleotides increases in number-worse in successive generations

Neutral mutations occur in regions that do not affect gene products or gene expression

• Loss of function is a mutation that reduces or eliminates function of the gene product

• Complete loss results from a null mutation

• Gain of function is a mutation that enhances function of gene product

• Could create a completely new function

Other terms related to mutations

Lethal mutations cause premature death of the organism

• Suppressor mutations mask the effect of an earlier mutation

• Mutation rate is the frequency with which a wild type allele

will undergo a mutation

• Mutations can either be spontaneous or induced

• Spontaneous mutations appear to occur naturally

• Induced mutations are associated with external factors

• Somatic cells have higher mutation rates than germ cells

causes of spontaneous mutations

Replication errors

• Occasionally, the wrong base is added during DNA replication

• Nitrogenous bases have different forms called tautomers

• One strand may not stay tightly paired to the complementary strand

• Called strand slippage or replication slippage

Nitrogenous bases can be chemically modified

• Formation of abasic sites is also called depurination

• Will lead to loss of a nucleotide pair during DNA replication

Nitrogenous bases can be chemically modified

• Deamination results in the loss of an exocyclic amine to change the purine or pyrimidine base

• Note: thymine cannot be deaminated

Causes of induced mutations

Induced mutations are caused by an external environmental factor

• Called a mutagen

• Examples of mutagens include multiple types of chemicals and radiation

• Ex. Radium is a naturally occurring radioactive element

• Decaying radium can excite fluorescent materials and make them glow

• When ingested, radium mimics calcium and accumulates in the bones

chemical mutagens

Base analogs-chemicals that substitute for a nucleotide

• Ex. 5-bromouracil

• Alkylating agents-chemicals that add an alkyl group to a nucleotide

• Ex. Mustard gases

Intercalating agents-chemicals that can slide between base pairs and distort the double helix

• Ex. Proflavin, acridine orange

• Adduct forming agents- chemicals that add a chemical moiety to DNA via a covalent bond

• Ex. Multiple environmental hazards

Radiation and mutagenesis

Two types of radiation can change DNA

• Ionizing radiation-removes an electron, X-rays and gamma rays

• Ultraviolet radiation

• Both either directly damage DNA or excite neighboring molecules that will cause the damage

UV radiation is associated with the formation of pyrimidine dimers

• Also called thymine dimers

• Covalent linkage of two adjacent pyrimidine bases

• Distorts shape of double helix

• Will cause cell death if damage is extensive

Ionizing radiation affects DNA in

multiple ways

• Excites stable molecules and creates free radicals (have an unpaired

electron)

• Free radicals can break phosphodiester bonds

multiple diseases are due to mutations in a single gene

Monogenic conditions are due to mutations in a single gene

• Sometimes called Mendelian diseases

• Complex diseases may have contributions from multiple genes

• Environment may also contribute to these diseases

Mechanisms of DNA repair

Homologous Recombination

• Non-homologous end joining

• Base excision repair (BER)

• Nucleotide excision repair

(NER)

• Mismatch repair (MMR)

*Some types of DNA damage

is repaired through simple

reversal using specific

enzymes*

repair of double-stranded breaks

Two mechanisms are used to repair

DSBs

• Homologous recombination

• Nonhomologous end joining (NHEJ)

• Goal is to reattach the broken

strands to each other

• Choice of which pathway used

depends on the availability of

homologous DNA

Nonhomologous end joinng ( NHEJ)

Here, there is no sister chromatid with homologous sequence

• If both ends are broken,

then how is correct sequence filled in?

• Ku proteins attach to broken ends and are recognized by additional factors

• Ends are trimmed and gap filled in

• more error prone

• occurs in G1

Base excision repair

Removal of an incorrect or damaged nucleotide

• Damage that does not significantly distort the shape of the double helix

• Corrects oxidative, deamination, alkylation, and abasic damage

• Repair is mediated through DNA glycolyases

Nucleotide excision repair

Removal of multiple nucleotides from the DNA

• Damage that does significantly distort the shape of the double helix

• Corrects pyrimidine dimers and DNA adducts

• Repair is mediated through DNA glycolyases

NER in the clinic

Xeroderma pigmentosum (XP)

• Greatly increases a patient’s sensitivity to sun exposure

• Severe sunburn with only a few minutes exposure

• Symptoms begin early in childhood

• Heritable, autosomal recessive

• Mutations are found in genes that make proteins for the NER pathway

Mismatch Repair

Corrects base mismatches and strand slippage loops that occur during replication

• Requires identification of the new strand and removal of multiple nucleotides surrounding mismatch

• MutSα heterodimer recognizes mismatch

• Exonuclease is recruited to remove portion of new DNA strand containing

mismatch

• Other proteins will stabilize parental strand while waiting for synthesis and ligation

MMR in the clinic

Lynch Syndrome

• Also known as hereditary non-polyposis colorectal cancer (HNPCC)

• Increased risk of developing multiple types of cancer, including:

• Colorectal cancer

• Endometrial cancer

• Stomach cancer

• Pancreatic cancer

• Etc.

• Heritable, autosomal dominant

• Mutations are found in genes that make proteins for the MMR

pathway

Transposable elements and mutation

Sequences of DNA that can move throughout & between chromosomes

• Vary in size

• Found in all organisms

• Prominent contributor to expansion of genome size

• Short repetitive sequences flank the mobile element

• Two categories

• DNA transposons

• Retrotransposons

DNA transposon

No RNA produced

• Move through the genome via “cut and paste”

• Inverted terminal repeat flanks the gene sequence for transposase

• Two types

• Autonomous

• Nonautonomous

Retrotransposon

Produce RNA as an intermediate

• Move through the genome as “copy and paste”

• Two types that can be either autonomous or nonautonomous

• Long terminal repeat (LTR)

• Non-long terminal repeat

• Contains gene sequence for reverse transcriptase

Multiple pathways to mutagenesis via transposition

Insertion into another portion of the genome is random

• Could disrupt reading frame, regulation of transcription, create splicing errors

• Depending on insertion, function of a gene product could be enhanced

• If two TEs are close to each other, this can lead to chromosomal rearrangements

Cancer is leading cause of death

Per the World Health Organization

(WHO):

• ~10 million deaths worldwide in

2020

• Most common types (worldwide)

are:

• Breast

• Lung

• Colorectal

• Prostate

• Skin

• Stomach

• Early detection and avoiding known

correlative & direct causes reduce

risk

Cancer is genetic disease

Cancer begins with genetic mutation and is defined by

overproliferation of somatic cells

• Mutations occur in genes that have a role in controlling the rate of

cell division

• Multiple mutations are usually found in cancerous cells

• Increase in cell number is due to three factors

• Cell growth and division at inappropriate rate

• Cell survival under conditions that usually trigger apoptosis

(programmed cell death)

• Telomere degeneration rules no longer apply

• *Cells that have all three factors usually show mutations in

multiple genes

types of mutations in cancer cells

Many different proteins (and genes that code for them) are needed to control

the rate of cell division

• In typical circumstances, cells cannot divide too quickly or too slowly

Proto-oncogenes

• Normal function is to promote progression through the cell cycle

• The gas pedal

• When mutated, they are called oncogenes and act in a dominant fashion

• Gain of function or loss of function?

DNA repair systems also require many proteins for proper function (and therefore

genes)

• If these proteins are defective, the initial mutations are not caught and fixed

• Mutations in these genes increase the likelihood of cells getting “stuck” with acquired mutations

Tumor suppressor genes

Tumor suppressor genes

• Normal function is to restrict

progression through the cell cycle

• The brake pedal

• Mutations act in a recessive manner,

so two copies of the mutation are

needed

• Gain of function or loss of function

Cyclin/CDK protein complexes

Cyclins are small proteins that regulate progression through the

stages of the cell cycle

• Mechanism of action is to associate with a kinase

• Cyclin dependent kinase (CDK)

• Can phosphorylate targets only when bound to a cyclin

Cyclin levels in cells oscillate with progression through the cycle

• Levels of CDK are constant no matter the stage of the cycle

Multiple different cyclins are present in cells

• These give specificity to the CDKs

• CDKs can only phosphorylate targets when bound to a cyclin, and the

specific cyclin tells them what those targets are

complexes act as a molecular on/off switches to move the cell from one phase of the cycle to another