BC-Monitor: Towards a Routinely Accessible Circulating Tumor DNA-Based Tool for Real-Time Monitoring Breast Cancer Progression and Treatment Effectiveness.

Circulating tumor DNA (ctDNA) is increasingly employed in the screening, follow-up, and monitoring of the continuously evolving tumor; however, most ctDNA assays validated for clinical use cannot maintain the right balance between sensitivity, coverage, sample requirements, time, and cost. Here, we report our BC-monitor, a simple, well-balanced ctDNA diagnostic approach using a gene panel significant in breast cancer and an optimized multiplex PCR-based NGS protocol capable of identifying allele variant frequencies below 1% in cell-free plasma DNA. We monitored a cohort of 45 breast cancer patients prospectively enrolled into our study receiving neoadjuvant chemotherapy or endocrine therapy or palliative therapy for metastatic diseases. Their tumor mutation status was examined in the archived tumor samples and plasma samples collected before and continuously during therapy. Traceable mutations of the used 38-plex NGS assay were found in approximately two-thirds of the patients. Importantly, we detected new pathogenic variants in follow-up plasma samples that were not detected in the primary tumor and baseline plasma samples. We proved that the BC-monitor can pre-indicate disease progression four-six months earlier than conventional methods. Our study highlights the need for well-designed ctDNA monitoring during treatment and follow-up, integrated into a real-time treatment assessment, which could provide information on the active tumor DNA released into the blood.

I-Block: a simple Escherichia coli-based assay for studying sequence-specific DNA binding of proteins.

We have developed a simple method called I-Block assay, which can detect sequence-specific binding of proteins to DNA in Escherichia coli. The method works by detecting competition between the protein of interest and RNA polymerase for binding to overlapping target sites in a plasmid-borne lacI promoter variant. The assay utilizes two plasmids and an E. coli host strain, from which the gene of the Lac repressor (lacI) has been deleted. One of the plasmids carries the lacI gene with a unique NheI restriction site created in the lacI promoter. The potential recognition sequences of the tested protein are inserted into the NheI site. Introduction of the plasmids into the E. coliΔlacI host represses the constitutive ß-galactosidase synthesis of the host bacterium. If the studied protein expressed from a compatible plasmid binds to its target site in the lacI promoter, it will interfere with lacI transcription and lead to increased ß-galactosidase activity. The method was tested with two zinc finger proteins, with the lambda phage cI857 repressor, and with CRISPR-dCas9 targeted to the lacI promoter. The I-Block assay was shown to work with standard liquid cultures, with cultures grown in microplate and with colonies on X-gal indicator plates.

DNA-dependent protease activity of human Spartan facilitates replication of DNA-protein crosslink-containing DNA.

Mutations in SPARTAN are associated with early onset hepatocellular carcinoma and progeroid features. A regulatory function of Spartan has been implicated in DNA damage tolerance pathways such as translesion synthesis, but the exact function of the protein remained unclear. Here, we reveal the role of human Spartan in facilitating replication of DNA-protein crosslink-containing DNA. We found that purified Spartan has a DNA-dependent protease activity degrading certain proteins bound to DNA. In concert, Spartan is required for direct DPC removal in vivo; we also show that the protease Spartan facilitates repair of formaldehyde-induced DNA-protein crosslinks in later phases of replication using the bromodeoxyuridin (BrdU) comet assay. Moreover, DNA fibre assay indicates that formaldehyde-induced replication stress dramatically decreases the speed of replication fork movement in Spartan-deficient cells, which accumulate in the G2/M cell cycle phase. Finally, epistasis analysis mapped these Spartan functions to the RAD6-RAD18 DNA damage tolerance pathway. Our results reveal that Spartan facilitates replication of DNA-protein crosslink-containing DNA enzymatically, as a protease, which may explain its role in preventing carcinogenesis and aging.