Nucleic Acids: DNA replication & Repair, Chromosome Organization by Dr Shin-ichiro Hiraga

The mechanism of DNA replication

DNA proofreading is a mechanism that corrects errors during DNA replication by removing and replacing incorrectly paired nucleotides

proofreading unit for editing on DNA polymerase- attaches to 3’ end of primer stand

the structure of the replication fork

Leading and lagging strands at replication forks due to the 5’ to 3’ direction of chain growth

lagging strand has okazaki fragments

topoisomerase I and II

enzymes that can alter the supercoiling of DNA by cutting one (type 1) or both stands of the 'DNA (type 2)

Type one allows one stand to untwist itself, and type 2 makes a way fo one double stranded DNA to pass through another double stranded DNA by cutting the latter.

topoisomerase inhibitors

Because dividing cells require greater topoisomerase activity due to increased DNA synthesis, topoisomerase inhibitors are used as chemotherapeutic agents

S phase

phase of the cell cycle where DNA is replicated

how cells select sites of replication initiation

origin recognition complex (ORC) binds to the origin of replication in G1, while Cyclin-dependent kinase (CDK) activity in S phase prevents re-replication by inhibiting the the assembly of pre-replicative complexes (pre-RCs) outside of the G1 phase. This ensures that DNA replication only occurs once per cell cycle.

Errors in origin replications

lead to overreplication of specific chromosomal regions. (gene amplification)

This seen commonly in cancer cells and can be an important prognostic indicator.

Such over-replication can also contribute to acquired drug resistance.

E.g. Methotrexate induces amplification of the Dihydrofolate Reductase locus

Diseases caused by defective licensing

Meier-Gorlin Syndrome (MGS)
• Caused by mutations in pre-RC genes, such as;
• ORC1, ORC4, ORC6, CDT1, or CDC6
• Patients have growth retardation, small ears, and patellar aplasia (= absence of kneecap)

how cells control whether and when to replicate DNA

In all cells studied, DNA replication is regulated by recruiting the replication machinery or “replisome” to sites called origins on the chromosome.

The replisome is a molecular machine that replicates the DNA bidirectionally from origins in a semiconservative fashion.

end replication problem

difficulty in replicating the ends of linear DNA molecules once the DNA prime is removed

The most common involves the use of an unusual reverse transcriptase, called telomerase

How telomerase solves the end replication problem

Telomerase is a reverse transcriptase carrying the template RNA. It functions by adding telomeric repeats to the ends of chromosomes, counteracting the shortening of telomeres that occurs during DNA replication.

Telomerase and Senescence (the process of growing old)

Telomerase is expressed in stem cells and germ line cells.
In contrast, in most somatic tissues, telomerase is expressed at very low levels or not at all -- as cells divide, telomeres shorten

Telomerase and Cancer

The presence of telomerase in cancer cells allows them to maintain telomere length while they proliferate

Studies in telomerase-deficient mice

suggest that it will probably depend on the type of tumor and exactly how that tumour maintains its telomeres

exons

Polypeptide-coding regions of the gene

introns

intervening DNA sequences
transcribed but non-coding
removed by splicing in the formation of the mature mRNA

regulatory sequences

specific segments of DNA that control the expression of genes, and doesn’t get transcribed

prokaryotic genome organisation

Genes are closely packed
• Few non-coding regions

Uninterrupted, i.e. lack introns
• DNA and mRNA are co-linear

Genes encoding proteins involved in the same metabolic pathway are often found in operons

operons

Clusters of genes are located in a contiguous array in the DNA
• Transcribed as a single unit from a single promoter

Polycistronic transcription

A continuous strand of mRNA that encodes a series of proteins. Cistron = Genetic unit encoding a single polypeptide

eukaryotic genome organision

The genome is structured into nucleosomes, which consist of DNA wrapped around histone proteins, forming chromatin.

The genes are often regulated by complex enhancer and silencer sequences far from the transcription start site and contain repetitive DNA sequences, transposable elements, and multiple origins of replication along the chromosomes

eukaryotic gene expression compared to prokaryotes

more regulated and complex

Satellite DNA

sections of DNA within an intron, telomere, or centromere that contain repeated DNA sequences. Satellite DNA is found in pericentromeric heterochromatin

repetitive DNA

noncoding DNA that can be associated with genes as regulatory sequences or as intervening (intron) sequences.

Some repeated DNA sequences are associated with human diseases, such as Huntington's disease, fragile X syndrome, and myotonic dystrophy

Chromosomes

Structures formed by tightly coiled and folded DNA, visible during cell division.

Nucleosomes

Basic units of chromatin formed when DNA wraps around histone proteins, with the DNA double helix encircling an octamer of histones.

Chromatin

A dynamic structure composed of coiled nucleosomes, involved in various DNA processes such as transcription, replication, recombination, and repair.

Chromatin looping

Mechanisms that facilitate the bringing together of enhancers and their target promoters.

DNA repair genes

Proteins present in cells that are responsible for repairing damaged DNA.

DNA-damage checkpoints

Checkpoints that halt the growth of damaged cells to facilitate DNA repair.

DNA repair pathways

Various pathways equipped with distinct enzymes to mend different types of DNA damage.

Nucleophagy

A natural mechanism of cellular cleaning that includes the protein TEX264.

Direct repair systems

Systems that repair nicks and rectify certain types of nucleotide modifications.

Consequences of unrepaired DNA damage

Three major outcomes

1. Cell death: The cell may undergo apoptosis if the damage is irreparable.

2. Cell-cycle arrest: The cell can enter a stable cell-cycle arrest state if the damage remains unaddressed.

3. Cancer: Damage to a gene encoding a DNA repair protein reduces the cell's ability to repair itself, potentially accumulating errors in other genes, which can result in cancer.