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The Holliday model

Posted by star on 2018-09-25 23:23:12
Hits:39

    The Holliday model postulated initiation of HR by a pair of single strand breaks (one in each duplex at identical sites), strand exchange to form symmetric heteroduplex DNA, and resolution of Holliday junctions to give either crossover or noncrossover outcomes. While this model explained some of the features of meiotic recombination in the fungi, it did not explain all of them nor did it explain some observations in other recombination systems. Foremost was the observation that recombination did not always result in a reciprocal exchange of a DNA segment to produce a symmetric pair of heteroduplexes. Instead, only one of the duplexes had a recombinant strand, resulting in the formation of an asymmetric heteroduplex. Another problem with the Holliday model was the need to introduce nicks in homologous duplexes at similar if not identical locations.

    Studies on RecA activity and nucleofilament formation on singie-stranded DNA Ied Matthew Meselson and Charles Radding to develop a new model in 1975. The Meselson-Radding model (sometimes called the Aviemore model because it was first proposed at a meeting in Aviemore, Scotland) retains some key features of the Holliday model, namely the formation of heteroduplex DNA as a HR intermediate, mismatch repair of the heteroduplex DNA to give 3:1 and 1: 3 gene conversion events, and the Holliday junction intermediate that could be resolved as noncrossover or crossover. The main features of the Meselson-Radding model are as follows:

    1. A single strand nick is introduced into a strand of one of the recombining duplexes.

    2. DNA polymerase extends the newly created 3'-end, causing the displacement of the strand on the other side of the nick. With the assistance of RecA, the displaced strand with a 5'-p tail invades a homologous region in the second duplex to form a D-loop. This invasion by the 5'-P tail does not fit with the functio......

An exchange protein RecA

Posted by star on 2018-09-25 23:19:08
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    E. coli uses several recombination pathways, and the RecA protein, It is required for all of them with the exception of a specialized RecE pathway. RecA binds to single-stranded DNA and double-stranded DNA as a helical filament.Binding of RecA to single-stranded DNA promotes the early steps of strand exchange between homologous DNA sequences. Approximately 6 to 8 RecA monomers are present in each helical turn, covering about 20 nucleotides and stretching the DNA by about 1. 5-fold.

    RecA promotes the pairing of single-stranded DNA with identical or very similar DNA sequences in vitro and in vivo. In vitro studies have shown that RecA binds to single-stranded DNA and promotes strand invasion to homologous sequences that can be in a single stranded form or a double stranded form. An important intermediate in strand invasion is the formation of the displacement loop (D-loop), which forms when one end of the invading strand displaces a strand with an identical or nearly identical sequence in double-stranded DNA. D-loop formation requires a free end for the invading strand. During strand exchange, ATP hydrolysis occurs through RecA activity. This hydrolysis promotes RecA dissociation from the single-strand DNA after strand exchange has occurred.

    The three stages of RecA-mediated strand exchange are

    1. Presynapsis. RecA binds to single-stranded DNA, forming a helical nucleofilament. Binding of RecA requires an ATP or dATP cofactor. Formation of the presynaptic complex is enhanced by the single-stranded DNA binding (SSB) protein, which helps by binding to single-stranded DNA and removing any secondary structure. This prepares the single-stranded DNA for association with RecA protein.

    2. Synapsis. During the synapsis stage, contacts are made between the RecA-coated single-stranded DNA and the double-stranded DNA, allowing the single-stranded DNA t......

New method for treating breast cancer

Posted by star on 2018-09-25 23:14:06
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    A cytokine signal in certain breast cancer stem cells can not only serve as a diagnostic tool for HER2-negative tumors, but also provide an effective therapeutic target.IL1b (a member of the interleukin 1 cytokine family) is often found in breast tumors, which may be an IL1 signal in women with HER2-negative metastatic breast cancer.The researchers found that the use of the natural IL1 receptor antagonist anakinra can effectively target this signal in patients.

    Anakinra has been widely used to treat autoimmune and autoinflammatory diseases, and researchers are also investigating its use as an adjuvant therapy to reduce inflammation in metastatic cancer, including metastatic colorectal cancer.
    The research results were recently published in Cancer Research,“IL-1 receptor antagonist controls transcriptional signature of inflammation in patients with metastatic breast cancer ”. In this new study, 11 women with advanced metastatic HER2-negative breast cancer received anakinra treatment, and the results showed that expression of IL1b and other related signaling factors decreased after two weeks of treatment.

    The researchers then treated patients with anakinra and standard chemotherapy for 4 months. The results showed that some patients experienced a decrease in pain during treatment and improved living treatment, and three of them were still alive.



Recombinant DNA phage

Posted by star on 2018-09-20 23:30:07
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Homologous recombination is not restricted to eukaryotic organisms. Viruses and bacteria also recombine their genomes. Studies of HR in these biological systems have provided important information about the physical nature and enzymology of HR. Recall from Chapter 8 that λ phages produce plaques on Escbericbia coli lawns. Plaque morphology is determined by several λ phage genes, which can be used for recombination studies.

Infection of E. coli with a pair of λ phages that differ in plaque morphology gives parental and recombinant plaque types. Matthew Meselson and Jean Weigle coinfected E. coli with a pair of λ phages that differed in genetic markers and in DNA density. One λ phage was used to infect bacterial cells cultured in a "light" medium that contained nutrients with normal isotopes 12C and 14N, while the other λ phage was used to infect bacterial cells cultured in a "heavy" medium that contained nutrients with the heavy isotopes 13C and 15N.

The λ phages released when the cells in each culture lysed were used to coinfect E. coli cultured in "light" medium. The cell lysate containing the progeny λ phages was centrifuged in a cesium chloride gradient, which separated the λ phages based on density. The λ phages were collected and then tested for parental or recombinant genotype. The major conclusion of this experiment was that recombinant DNA can be formed by breakage and rejoining as indicated by the appearance of genetic recombinants that had both heavy and light DNA.



Homologous recombination repairing DNA

Posted by star on 2018-09-20 18:48:45
Hits:33

    Homologous recombination not only forms the basis for much of the genetic diversity among progeny of common parentage, but it is also essential for correct segregation of homologous chromosome pairs at the first meiotic division. When HR does not occur, homologous chromosomes are not held together and therefore segregate randomly at meiosis I, giving rise to meiotic products that are missing chromosomes or have extra copies of chromosomes. Such grossly aneuploid gametes are not functional. In somatic cells, HR is needed to repair double-strand breaks that arise from exogenous DNA damage sources such as x-rays and from endogenous sources such as defective DNA topoisomerases. Homologous recombination can also be used to repair single-stranded DNA gaps at replication forks. Homologous recombination can restart replication forks that have stalled at a lesion on the template DNA strand or can reinitiate a replication fork that has collapsed at a nick or other single strand interruption on the template DNA strand. Last, HR is needed to maintain telomeres, the specialized ends of chromosomes, when the enzyme telomerase that normally takes care of replicating telomeres is missing.

    In the early days of genetics after the work of Gregor Mendel was rediscovered, two observations led to the concept of linkage the idea that genes can be genetically and physically linked and hence will not segregate randomly from each other during the first division of meiosis.

    Reginald Punnett indicating that two sweet pea traits tended to segregate with each other more often than expected by random segregation. This phenomenon was called coupling. However linkage was not complete, and sometimes nonparental meiotic products or gametes were recovered, These were called nonparental or recombinant gametes and were the product of meiotic crossing over or recombination. The term crossing over was coined by Thomas Hunt Morgan.......

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