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Chang, Seungwoo

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Chang

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Seungwoo

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Chang, Seungwoo

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2016 - 20172

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Author's Publications: search.filters.isAuthorOfPublication.a4f7d8b3-59ad-4756-8502-b6ad21ff6b3e

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    Mapping DNA polymerase errors by single-molecule sequencing
    (Oxford University Press, 2016) Lee, David; Lu, Jenny; Chang, Seungwoo; Loparo, Joseph; Xie, Xiaoliang
    Genomic integrity is compromised by DNA polymerase replication errors, which occur in a sequence-dependent manner across the genome. Accurate and complete quantification of a DNA polymerase's error spectrum is challenging because errors are rare and difficult to detect. We report a high-throughput sequencing assay to map in vitro DNA replication errors at the single-molecule level. Unlike previous methods, our assay is able to rapidly detect a large number of polymerase errors at base resolution over any template substrate without quantification bias. To overcome the high error rate of high-throughput sequencing, our assay uses a barcoding strategy in which each replication product is tagged with a unique nucleotide sequence before amplification. This allows multiple sequencing reads of the same product to be compared so that sequencing errors can be found and removed. We demonstrate the ability of our assay to characterize the average error rate, error hotspots and lesion bypass fidelity of several DNA polymerases.
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    Single-molecule imaging reveals multiple pathways for the recruitment of translesion polymerases after DNA damage
    (Nature Publishing Group UK, 2017) Thrall, Elizabeth; Kath, James E.; Chang, Seungwoo; Loparo, Joseph
    Unrepaired DNA lesions are a potent block to replication, leading to replication fork collapse, double-strand DNA breaks, and cell death. Error-prone polymerases overcome this blockade by synthesizing past DNA lesions in a process called translesion synthesis (TLS), but how TLS polymerases gain access to the DNA template remains poorly understood. In this study, we use particle-tracking PALM to image live Escherichia coli cells containing a functional fusion of the endogenous copy of Pol IV to the photoactivatable fluorescent protein PAmCherry. We find that Pol IV is strongly enriched near sites of replication only upon DNA damage. Surprisingly, we find that the mechanism of Pol IV recruitment is dependent on the type of DNA lesion, and that interactions with proteins other than the processivity factor β play a role under certain conditions. Collectively, these results suggest that multiple interactions, influenced by lesion identity, recruit Pol IV to sites of DNA damage.