CHROMOSOMAL DNA REPLICATION

Strands of duplex DNA are antiparallel A pre-existing nucleic acid strand is copied by rules of Watson-Crick base pairing Figure 5-3 Molecular Biology of the Cell (© Garland Science 2008)

DNA polymerase requires a 3’-OH to synthesize DNA Figure 5-4 Molecular Biology of the Cell (© Garland Science 2008)

Direction of DNA synthesis is always 5’  3’ Because strands are antiparallel, this requires a special growing fork: one strand synthesized continuously; the other strand synthesized discontinuously Figure 5-7 Molecular Biology of the Cell (© Garland Science 2008)

DNA replication is bi-directional Two forks move in opposite directions from one origin Figure 5-25 Molecular Biology of the Cell (© Garland Science 2008)

NUCLEIC ACID SYNTHESIS IS GOVERNED BY FOUR IMPORTANT CHARACTERISTICS: A pre-existing nucleic acid strand is copied by rules of Watson-Crick base pairing Nucleic acid strands grow in only one direction, 5’3’ Polymerases synthesize nucleic acids Duplex DNA synthesis requires a special growing fork because the strands are antiparallel

DNA Replication: Step-by-Step

DNA REPLICATION UTILIZES SIX SPECIALIZED MECHANISMS: Initiation Unwinding Priming Unidirectional fork movement Untangling Termination

Initiation begins at origins of replication

Helicase in an allosteric motor protein that unwinds DNA Figure 5-14 Molecular Biology of the Cell (© Garland Science 2008)

Single-strand binding proteins (SSB) prevent reannealing without preventing base pairing Figure 5-16 Molecular Biology of the Cell (© Garland Science 2008)

DNA synthesis requires an RNA primer Figure 5-11 Molecular Biology of the Cell (© Garland Science 2008)

A regulated sliding clamp permits processive DNA synthesis on the leading strand and rapid reassembly of the replication complex on the lagging strand

The RNA primer is removed and replaced by DNA Figure 5-12 Molecular Biology of the Cell (© Garland Science 2008)

Ligase synthesizes the phosphodiester bond that links Okazaki fragments Figure 5-13 Molecular Biology of the Cell (© Garland Science 2008)

DNA polymerase proofreads as it synthesizes DNA Figure 5-8 Molecular Biology of the Cell (© Garland Science 2008)

Figure 5-9 Molecular Biology of the Cell (© Garland Science 2008)

Table 5-1 Molecular Biology of the Cell (© Garland Science 2008)

Germline mutations affecting the DNA polymerase (E and D) proofreading domains predispose to colorectal adenomas and carcinomas Figure from Seshagiri commentary Nature Genetics 45:121, 2013 on: Palles et al, Nature Genetics 45:136, 2013

The replication proteins act together as one large multienzyme complex requiring the lagging strand to fold back Figure 5-19a Molecular Biology of the Cell (© Garland Science 2008)

The winding problem that arises during DNA replication

DNA topoisomerase I relieves supercoiling ahead of the replication fork Figure 5-22 Molecular Biology of the Cell (© Garland Science 2008)

DNA topoisomerase II untangles DNA after it is replicated Figure 5-24 Molecular Biology of the Cell (© Garland Science 2008)

Effective class of cancer chemotherapeutic drugs: DNA topoisomerase II inhibitors: Increase stability of topoII-DNA cleavage complexes High levels of transient DNA breaks Become permanent double-stranded breaks when replication machinery or helicases attempt to traverse DNA:topoII block in DNA Too many breaks: cell death Figure 5-24 Molecular Biology of the Cell (© Garland Science 2008)

DNA topoisomerase II inhibitors demonstrate some anti-cancer activity as single agents, but are now most often used in combination therapies

As a result of treatment with DNA topoisomerase II-targeting drugs, some patients develop chromosome rearrangements: MLL gene fusions which cause leukemia

DNA REPLICATION UTILIZES SIX SPECIALIZED MECHANISMS: Initiation Unwinding Priming Unidirectional fork movement Untangling Termination

…short break, then: Comparing Prokaryotic and Eukaryotic DNA Replication: Features Unique to Eukaryotes

Prokaryote: One origin Can initiate replication before previous cycle completed (after a short lag period) as long as sufficient nutrients Figure 5-26 Molecular Biology of the Cell (© Garland Science 2008)

Eukaryotic DNA replication occurs once during the cell cycle

Studying eukaryotic origins of replication Figure 5-29 Molecular Biology of the Cell (© Garland Science 2008)

Each eukaryotic chromosome has many replication origins Figure 5-34 Molecular Biology of the Cell (© Garland Science 2008)

What determines whether a chromosome region will be replicated early vs. later during S phase? Chromatin structure Heterochromatin later than euchromatin Gene density Gene-dense regions replicate earlier than gene-poor regions Gene expression Regions with highly expressed genes in a given tissue replicate earlier than gene regions that are not expressed or expressed at a lower level in that tissue

Eukaryotic Control of Replication: Preventing Re-Replication and Coordinating Replication and Cell Division

Mechanism to ensure each origin is activated only once per cell cycle

Cell controls assure that each region of eukaryotic DNA is usually replicated only once and that all DNA is replicated before the onset of mitosis.

Patterns of histone modification can be inherited

Figure 5-39 Molecular Biology of the Cell (© Garland Science 2008)

Models for inheritance of heterochromatin during DNA replication Figure 4-51 Molecular Biology of the Cell (© Garland Science 2008)

Figure 4-52 Molecular Biology of the Cell (© Garland Science 2008)

EUKARYOTIC CHROMOSOMAL DNA REPLICATION DIFFERS FROM PROKARYOTIC DNA REPLICATION IN THREE WAYS: It is compartmentalized within the nucleus, partitioning it from the site of synthesis of replication proteins and precursors and from extracellular stimuli that may trigger initiation of synthesis; It begins at multiple origins, activated throughout S-phase in a precise and temporally regulated manner; The nucleosomal proteins must be duplicated along with the DNA to maintain proper chromosomal organization.

The “End-Replication” Problem: Role of Telomeres

Figure 4.17. Textbook of Biochemistry, 5th Edition. The end-replication problem: how to synthesize DNA complementary to the very end of the linear chromosome on the lagging strand The 3’ end of the template DNA strand has no room for a primase to synthesize a primer.

Telomerase is not expressed in all cells. Telomerase is a riboprotein containing an RNA template for synthesizing the G-rich telomere sequence Telomerase is not expressed in all cells. Primarily expressed in germ cells, stem cells and cancer cells Figure 5-40 Molecular Biology of the Cell (© Garland Science 2008)

The 3’ end of the template DNA strand has no room for a primase to synthesize a primer. Telomere synthesis elongates the 3’ ends Figure 5-41 Molecular Biology of the Cell (© Garland Science 2008)

Telomere shortening can trigger replicative senescence in human cells Telomere ends are protected from degradation and repair by a unique folded structure Telomere shortening can trigger replicative senescence in human cells Figure 5-42 Molecular Biology of the Cell (© Garland Science 2008)

Telomeres function to protect single-stranded chromosomal ends from recombination, fusion, and from being recognized as damaged DNA. Telomere shortening can trigger replicative senescence in human cells that don’t express telomerase.

Example of the Clinical Consequences of Faulty DNA Replication: the Nucleotide Repeat Expansion Diseases

Model for trinucleotide repeat expansion: DNA replication slippage repeat sequences Polymerase pauses while replicating repeat tract Polymerase transiently dissociates During reassociation, is misalignment of template and nascent strand

Hallmark of trinucleotide expansion diseases: Classification of the trinucleotide repeat, and resulting disease status, depends on the number of CAG repeats (poly Q: glutamine) in Huntington’s Disease Repeat count Classification Disease status <28 Normal Unaffected 28–35 Intermediate 36–40 Reduced Penetrance +/- Affected >40 Full Penetrance Affected Hallmark of trinucleotide expansion diseases: Tendency of the tandem arrays to increase in size from generation to generation. Anticipation: progressive increase is disease severity and decrease in age of onset seen with successive generations in an affected family

Many examples of trinucleotide expansion diseases: Trinucleotide expansion can have different effects on gene transcription, translation, or stability of protein product, depending on location of triplet repeats within the relevant gene Many copies Few copies

Slipped mispairing during DNA replication is a likely mechanism for triplet repeat expansion. The wild-type genes associated with triplet repeat disorders contain variable but relatively low numbers of tandem (adjacent) trinucleotide units. Individuals with genes carrying repeats above a threshold number are affected. Trinucleotide repeat expansion diseases demonstrate anticipation